Transplant. Rev. (1972), Vol.

13 35-66
Published by Munksgaard, Copenhagen, Denniark
No part may be reproduced by any process without written permission from the author(s)

The Relationship Between Humoral and

Cell-Mediated Immunity
C. R. PARISH

It has been known for many years that when an antigen is injected into an
animal, both humoral and cell-mediated immunity can be induced. However,
different antigens show a remarkable variation in their capacity to induce
these two immune responses. For example, some antigens appear to be pre-
disposed to induce antibody formation, whilst other antigens predominantly
provoke delayed-type hypersensitivity (Turk 1967). Furthermore, the route
and mode of injection of any one antigen can have a profound effect on the
type of immune response which occurs (Pappenheimer & Freund 1959). The
biological factors which determine whether humoral or cell-mediated im-
munity predominates are not well understood, although it has been tenta-
tively proposed that delayed-type hypersensitivity is induced by antigens
which are only partially recognized as 'foreign' by the responding animal
(Pearson & Raffel 1971). This review will attempt to throw some light on
the factors which modulate humoral and cell-mediated immunity in vivo,
and in particular, to present compelling evidence that in many situations
the humoral and cellular responses are mutually antagonistic.

I, EVIDENCE FOR AN INVERSE RELATIONSHIP

BETWEEN HUMORAL AND CELL-MEDIATED IMMUNITY

In recent years there have been numerous reports in the literature that
selective stimulation or suppression of either delayed-type hypersensitivity
or antibody formation can be achieved. Probably the most striking example
of this phenomenon was the observation reported by many workers (Battisto
& Miller 1962, Asherson & Stone 1965, Dvorak et al. 1965, Borel et al.

Department of Microbiology, John Curtin School of Medical Research, Australian

National University, Canberra, A.C.T., 2600, Australia,
36 C. R. PARISH

1966, Crowle & Hu 1966, Loewi et d. 1966) that pretreatmeat of adult

animals with antigen in saline can suppress delayed hypersensitivity and that
this suppression is usually accompanied by 'control' and sometimes by
heightened levels of antibody. This phenomenon has been referred to
variously as 'immune deviation' (Asherson & Stone 1965), 'split tolerance'
(Crowle & Hu 1966) and 'preimmunization tolerance' (Loewi et cd. 1966).
On the other hand, it has been observed that antibody tolerance to tuberculin
(Janicki et al. 1970) and lysozyme (Thompson et al. 1972) in adult animals
was accompanied by normal levels of delayed-type hypersensitivity. Further-
more, it has frequently been reported that low doses of antigen preferentially
induce cell-mediated immunity (e.g. Salvin 1958). These studies led to the
belief that humoral and cell-mediated immunity represent completely inde-
pendent immunological responses and might evolve from separate cell
lineages. The recent demonstration that cellular immune reactions are
mediated by thymus-dependent T cells, whereas thymus-independent B cells
synthesize antibody, supports this concept. However, there is now ample
evidence that interaction between T and B cells is frequently essential for
antibody production (Miller & Mitchell 1969, Davies 1969, Qaman &
Chaperon 1969, Taylor 1969). Several lines of experimental evidence
recently accumulated in our laboratory will now be presented which strongly
suggest that, in fact, an inverse relationship often exists between delayed-
type hypersensitivity and antibody formation.

A. Studies with Chemically Modified Antigens

Perhaps the most compelling evidence that an inverse relationship exists
between humoral and cell-mediated immunity has been obtained with
chemically modified Salmonella adelmde flagellin (Parish I971a,b,c). By
acetoacetylating the flagellin molecule to varying extents, a series of flagellin
preparations were obtained which had slightly to greatly reduced antigenic
activities. This loss in antigenic activity was measured accurately by quanti-
tative microprecipitin-inhibition studies and allowed a direct comparison of
antigenic activity and immunogenicity. From the shapes of the inhibition
curves, particularly with the heavily acetoacetylated preparations, it appeared
likely that acetoacetylation reduced the affinity of all antigenic determinants
in the molecule rather than selectively destroying antigenic specificities
(Parish 1971b). It is probable that acetoacetylation modifies antigenicity
by directly substituting antigenic groupings and by indirectly altering the
overall conformation of the flagellin molecule, particularly by changing
the net charge of the protein.
The immunological properties of the acetoacetylated flagellins are sum-
 HUMORAL AND CELLULAR IMMUNITY 37

marized in Figures 1-3. From these results the following observations can
be made and conclusions drawn:-
(a) As the antigenic activity of flagellin was reduced by acetoacetylation
there was a rapid decline in the ability of the molecule to initiate antibody
formation but an enhanced capacity of the protein to induce flagellin-specific
delayed-type hypersensitivity. This phenomenon occurred when the aceto-
acetyl-flagellins were injected into either unprimed (Figure 1) or flagellin
primed (Figure 2) animals. Both these experimental systems strongly suggest
an inverse relationship between cell-mediated immunity and humoral anti-
body responsiveness.
(b) Not only did acetoacetylation convert flagellin from an antigen which
predominantly induced antibody formation to an antigen which exclusively
provoked cellular immunity, but also the acetoacetyl-flagellins very efficiently
induced flagellin-specific antibody tolerance (Figure 3). However, such
tolerance was induced only in unprimed rats and not in flagellin primed
animals, presumably because primed individuals possessed a larger pool of

90

80
X
TO I
o

z
so I
i-O <
0.
5o
30 "-

RELATIVE AMIICENIC 4C1IV1TY

Figure 1. The relationship between the antigenic activity of the aceloacetyl derivatives
of flagellin and their ability to induce a primary antibody response ( • • ) and
delayed-type hypersensitivity (Q D) "> flagellin in rats. Antibody titers and
delayed-type hypersensitivity were measured 35 days after the injection of 1 /^g
of the various acetoacetyi-flagellins in FCA. The hypwrsensitivity reactions were
elicited in the hind footpads with 1 fig of flagellin in saline and footpad swellings
were determined 24 hr after elicitation. Relative antigenic activity = the antigenic
activity of the acetoacetyl-flagellins compared with unmodified flagellin. Vertical
bars represent standard errors of the means. (Adapted from Parish 1971c).
38 C. R. PARISH

immunocompetent cells. This interpretation was further confirmed by the

observation that acetoacetyl-flagcUin induced antibody tolerance much less
efficiently in a high responder rat strain than in a low responder strain
(Parish & Uew 1972, Parish 1972b).
(c) Very extensive acetoacetylation of flagellin resulted in a preparation with
such reduced antigenicity that it began to become immunologically unrecog-
nizable as flagellin (see Figures 1 and 3).
(d) There was no evidence that acetoacetylation converted flagellin into a
new antigen which was capable of producing antibodies and delayed hyper-
sensitivity with a completely different spectrum of specificities. No antibodies
or cellular immunity against the acetoacetyl group could be detected and the

VO WJO"' 1x10*' 1.10"^ tdO"' lilO'*

RELATIVE ANTIGENtC ACTIVITV

Figure 2. The relationship between the antigenic activity of the acetoacetyl derivatives
of flagellin and their ability to induce delayed-type hypersensitivity (Q • ) and
trigger a secondary antibody response ( • • ) to flagellin. Rats were primed with
1 fig of unmodified flagellin in saline and 28 days later challenged with 1 /ag of the
various acetoacetyl-flagellins in saline. Delayed-type hypersensitivity was elicited 14
days post-challenge by the injection of 1 ^g of flagellin in saline into the hind
footpads. The delayed responses represent the means of the 24, 48 and 72 hr footpad
swelling responses. The antibody titers represent the means of the 7- and 14-day
secondary antibody titers. The dotted line ( ) represents the antibody and
delayed-type hypersensitivity responses of control rats which were primed with 1 fig
of flagellin but challenged with saline alone. Relative antigenic activity =: the anti-
genic activity of the acetoacetyl-flagellins compared with unmodified flagellin. Vertical
bars represent standard errors of the means. (Reproduced from Parish 1^72b, copyright
Academic Press, New York).
 HUMORAL AND CELLULAR IMMUNITY 39

delayed hypersensitivity induced by the acetoacetyl-flagellins was most ef-

ficiently elicited by unmodified flagellin (Parish 1971c).
One of the most remarkable features of this study was the dramatic
change in the immunogenicity of flagellin following only mitior changes in
antigenic activity. For example, only a 50 per cent reduction in the anti-
genicity of flagellin (as measured by the microprecipitin-inhibition test)
almost completely destroyed the antibody forming capacity of the protein
and substantially enhanced the delayed response induced. In contrast,
delayed-type hypersensitivity and antibody tolerance to flagellin was induced
by acetoacetylated flagellins which had drastically reduced antigenicities-
This apparent divergence in the specificity requirements for humoral and
cell-mediated immunity will be discussed in greater detail in a later section
of this review.
Preliminary studies have indicated that chemical moieties other than the
acetoacetyl group can have similar effects on the immunological properties

102^0

3560

J60

10 1-10- 1.10" .10"* Z ^ ^.in-"

REIATIVE ANT/GENIC ACTIVIiy

Figure 3. The relationship between the antigenic activity of the acetoacetyl derivatives
of flagellin and their ability to induce antibody tolerance to flagellin. Legend: 28-day
antibody titers to flagellin of rats which had been pretreated with the various
acetoacetyl-flagellins {1 fig in FCA) prior to flagellin challenge (1 ug in saline)
(^ ^ ) ; 28-day antibody response of control rats which had been pretreated with
FCA and then challenged with 1 fig of flagellin in saline ( ). Relative antigenic
activity = the antigsnic activity of the acetoacetyl-flagellins compared with unmodified
flageliin. Vertical bars represent standard errors of the means. (Reproduced from
Parish 1971c).
40 C. R. PARISH

of flagellin (M. Venning, in preparation). However, it appears that only

chemical groupings with a size resembling that of the acetoacetyl group
are effective. Bulkier groups seem to ohliterate both the antibody and
cellular immune responses to flagellin. A more detailed investigation of the
immunological effects of a range of chemical substituents is currently under
investigation.
A diversion of the immune response from antibody formation into delayed
hypersensitivity was observed in this laboratory when other antigens were
chemically modified. Probably the most clear cut example was with sheep
erythrocytes, where a combination of periodate oxidation and acetoacetyla-
tion converted this antigen from a form which predominantly induced
humoral antibodies to a form which exclusively provoked cell-mediated
immunity (Parish 1972a). Again, an apparent inverse relationship be-
tween humoral and cell-mediated immunity was evident. Acetoacetylated
preparations of hemocyanin, fowl ^--globulin and nematode allergen (S.
Hogarth-Scott, personal communication) have also induced enhanced delayed
hypersensitivity responses.
Several reports from other laboratories have suggested that chemically
modified antigens express altered immunological properties. Benacerraf &
Gell (1959) reported that a range of protein antigens substituted with either
picryl, acetyl, or ethoxymethylene-phenyloxazolone groups tended to induce
protein specific delayed-type hypersensitivity more effectively than the
native protein. In contrast, no antibodies against the protein carrier could
be detected, although antibody was produced against the haptenic groupings.
Furthermore, it has been demonstrated recently tbat carboxymethylated
Iysozyme, although producing no detectable antibodies against lysozyme,
can induce both delayed-type hypersensitivity and antibody tolerance to
lysozyme (Thompson et al. 1972). Related to these findings is the dem-
onstration that heat-denatured proteins are as effective as native proteins
in provoking delayed hypersensitivity, in spite of the fact that denaturation
greatly modifies their antigenicity (Gell & Benacerraf 1959). Similar results
were obtained with flagellin which had been denatured and partially
hydrolyzed with sodium hydroxide (M. Veiming, unpublished).
Thus, it appears that chemical modification of antigens via a range of
substitution techniques represents a simple and effective means of obtaining
antigenic preparations which exclusively provoke cell-mediated immunity
and, under certain circumstances, induce antibody tolerance. The theoretical
and practical implications of these procedures will be discussed in subsequent
sections of this review.
 HUMORAL AND CELLULAR IMMUNITY 41

B. Antibody Tolerance Studies

It was originally demonstrated by Mitchison (1964) and Dresser (1962a,b)
that antibody tolerance could be induced in adtilt animals by injecting
soluble antigens at two different dose levels. Intermediate doses of antigen
usually resulted in etihanced antibody titres rather than in antibody tolerance.
These two regions of antibody tolerance have been termed 'high zone' and
'low zone' tolerance according to the relative amounts of antigen required
to induce the tolerant state. It now appears that high and low zone antibody
tolerance can be induced by a wide range of antigens (Dresser & Mitchison
1968).
It has been known for several years that high and low zone antibody
tolerance to 5. adelaide flagellin can be induced in adult rats by multiple
injections of a cyanogen bromide (CNBr) digest of flagellin at two widely
spaced dose levels (Ada & Parish 1968). The levels of cell-mediated im-
munity existing during these tolerant states were recently reported (Parish
& Liew 1972). These studies are summarized in Figure 4. It can be seen
that both high and low zone antibody tolerance to flagellin were accompanied

I,:BO

DAILY DOSE INJECTED

Figure 4. Antibody titers (I I) and delayed-type hypersensitivity responses

(O D) of strain W Wistar rats injected intraperitoneally daily for 28 days with
varying amounts of the cyanogen bromide (CNBr) digest of flagellin and then
challenged with 100 n% of flagellin in saline. The antibody titers represent the msan
of the 7-, 14-, 21- and 28-day post challenge titers. Delayed-type hypersensitivity was
elicited 28 days after the flagellin challenge (represents 24-hour footpad swellings).
The dotted line ( ) represents the antibody and delayed hypersensitivity responses
of control rats which were injected only with 100 ^g of flagellin in saline. Vertical
bars represent standard errors of the means (Reproduced from Parish &. Liew 1972).
42 C. R. PARISH

by enhanced levels of delayed-type hypersensitivity. Conversely, the en-

hanced antibody responses induced by intermediate doses of the CNBr
digest were associated with suppressed cell-mediated immunity (i.e. 'cellular
immunity tolerance'). This system provides further evidence for a possible
'mirror image' relationship between humoral and cell-mediated immunity.

C. Studies with Passively Administered Antibody

There is ample experimental evidence which indicates that passively ad-
ministered antibody can suppress antibody formation both in vivo (Uhr &
Moller 1968) and in vitro (Land et al, 1969, Pierce 1969, Feldmann &
Diener 1970). In contrast, the effect of passive antibody on cell-mediated
immunity to soluble antigens has been relatively neglected, although there
have been numerous reports that passive antibody can readily suppress the
induction of cell-mediated immunity against tissue ^afts (i.e-, produce
immunological enhancement) (Kaliss 1958, Snell et al. 1960, Moller &
Moller 1966, Uhr & Moller 1968). Recently we have investigated the effect
of passively administered antibody on the humoral and cell-mediated im-
munity induced by systemically injected antigens and observed that sup-
pression of antibody formation was frequently accompanied by enhanced
cell-mediated immunity (Liew & Parish 1972a). This phenomenon was
observed with soluble (flagellin), particulate (polymerized flagellin), and
cellular (sheep erythrocytes) antigens and was also obtained with both
homologous and heterologous passive antibody. Figure 5 depicts the effect
of passively administered anti-polymerized flagellin antibody on the subse-
quent humoral and cell-mediated immune responses to polymerized flagellin.
It is clearly evident that suppression of antibody formation was associated
with augmented levels of delayed-type hypersensitivity. This experimental
system again suggests that antibody formation and cell-mediated immunity
may well be opposing immunological processes in adult animals.

It should be noted that very high concentrations of passive antibody were

capable of suppressing both humoral and cell-mediated immunity (Liew
& Parish 1972a). Similar results have been reported by other workers
(Crowle & Hu 1965, 1968, Axeh-ad 1968). These results suggest that very
high concentrations of passive antibody simply mask antigenic determinants
and prevent these determinants from reacting with receptors on immuno-
competent cells (i.e., a 'peripheral effect') (Uhr & Moller 1968). In contrast,
lower concentrations of passive antibody had a 'central effect' on the im-
mune system (Rowley & Fitch 1964, Feldmann & Diener 1970) by diverting
the immune response into cell-mediated immunity rather than antibody
formation. It has subsequently been shown that very low concentrations of
 HUMORAL AND CELLULAR IMMUNITY 43

passive antibody can actually enhance the antibody response to soluble

(flagellin) and cellular (sheep erythrocyte) antigens (Liew •&. Parish 1972b).
In the case of flagellin, the heightened antibody response was accompanied
by suppressed delayed-type hypersensitivity. These results will be discussed
in greater detail later.

i.O

03.0
o

160r

2 10 50 250
CONCfNTRATION OF ANTIBODY

Figure 5. Comparison of the effect of different concentrations of passively administered

guinea pig anti-polymerized flagellin antibody on the humoral and cell-mediated
immune responses to piolymerized flagellin. Groups of rats were injected intraperi-
tonealiy with 1.0 ml of the various antiserum dilutions. One hour later, each rat
received intraperitoneally 0.5 ixg of polymerized flagellin in 0.5 ml saline. On day
28, delayed-type hypersensitivity was elicited in the hind footpads by the injection
of 0.5 fig of flagellin in saline. Antibody responses represent the mean of the 7-,
]4-, 21- and 28-day antibody titers, the levels of serum antibtKiy due to passive im-
munization being subtracted. Cell-mediated immunity is expressed as the 24-hour
footpad swelling. Vertical bars represent standard errors of the means. (Reproduced
from Liew & Parish 1972a, copyright Academic Press, New York).
44 C. R. PARISH

D. Additional Comments
So far in this section evidence has been presented which strongly suggests
the existence of an inverse relationship betvi^een humoral and cell-mediated
immunity. It should be emphasized, however, that although this phenomenon
has occurred in the majority of systems studied, there have been some
partial exceptions. For example, one strain of rat was found which exhibited
only a partial inverse relationship when injected with multiple doses of the
cyanogen bromide digest of flagellin (Parish & Liew 1972). Also, in some
experiments with polymerized flagellin it was observed that substantially
lower concentrations of passive antibody were required to enhance delayed
hypersensitivity than to suppress primary antibody formation (Liew &
Parish 1972b). These apparent discrepancies could be due to the assay
systems used, as antibody titre may not be a true reflection of the number
of antibody forming cells, and the relative sensitivities of antibody and
delayed-type hypersensitivity measurements are not known.
From the data presented in this review it could be argued that the inverse
relationship between humoral and cell-mediated immunity merely represents
a masking of delayed-type hypersensitivity by serum antibody, antibody
blocking the delayed response by competing with hypersensitivity cells for
antigen. There are several lines of evidence which suggest that this is not
the case. Firstly, the passive administration of specific hyperimmune serum
into hypersensitive animals has always failed to mask delayed-type hyper-
sensitivity (Parish 1971c). Secondly, peritoneal cells from rats expressing no
delayed-type hypersensitivity but high antibody titres were unable to transfer
delayed responsiveness to normal recipients (Parish 1971c, Liew & Parish
1972a). On the other hand, peritoneal cells from hypersensitive animals
very readily transferred delayed reactivity (Parish 1971c, 1972b, Liew &
Parish 1972a). Finally, the acetoacetylated flagellins induced and expressed
comparable levels of delayed-type hypersensitivity when injected into either
normal rats or into rats which were already primed to flagellin and ex-
pressing substantial antibody titres (Table II, Parish 1972b).
One additional factor which should be noted is that delayed-type hyper-
sensitivity to many antigens can be partially to completely desensitized by
the injection of as little as 1-20 jug of antigen (Uhr & Pappenheimer 1958,
Parish & Liew 1972). This may explain why the striking inverse relationship
between humoral and cell-mediated immunity observed with flagellin has
not been reported previously with other antigens, as in most other systems
large quantities of slowly eliminated antigen are needed to immunize and
therefore desensitization could easily occur. Flagellin has the advantage that
it is very rapidly eliminated from the circulation and only minute amounts
of antigen are needed to immunize (Nossal et al. 1964).
 HUMORAL AND CELLULAR IMMUNITY 45

II. POSSIBLE MECHANISMS REGULATING HUMORAL AND

CELL-MEDIATED IMMUNITY IN VIVO

A. Cireulating Antibody
The finding by several workers that passively administered antibody given
subsequent to antigenic stimulus can suppress antibody formation has led
to the suggestion that actively formed serum antibody may have a regulatory
role (Uhr & Baumann 1961, MoUer & Wigzell 1965, Morris & Moller
1968). It now seems apparent that an inverse relationship exists between
humoral and cell-mediated immunity (see earlier) and that, depending on
the circumstances, circulating antibody can actively divert the immune
response into either one or other of these states (Liew & Parish 1972a,b,c).
In fact, such factors as the physical nature of the antigen injected, the level
of circulating antibody, and the class of passive antibody administered
appear to have a profound effect on the type of immune reaction which
is induced. Table I summarizes the effect of these factors on humoral and
cell-mediated immunity.
It was fouad that high levels of passive antibody almost invariably
enhanced the induction of delayed-type hypersensitivity and suppressed anti-
body formation, irrespective of the type of antigen or class of antibody
injected. This phenomenon may represent an important 'feedback mecha-
nism' in vivo since, to effectively combat many infections, it is probably
desirable that animals express high levels of both humoral and cell-mediated
immunity. Thus it appears that antibody not only actively feeds back on
its own synthesis but can actually divert the immune response into a com-
pletely different form, namely cell-mediated immunity. In contrast, the effect
of intermediate and low concentrations of antibody was highly dependent
upon the class of antibody and type of antigen injected (Liew & Parish
1972b). For example, at intermediate concentrations and with either flagellin
or sheep erythrocytes as antigen, it was found that different classes of passive
antibody varied in their capacity to suppress antibody formation and enhance
cell-mediated immunity, being in the order: yl ^ y\ = IgM- On the other
hand, all three classes of antibody had similar immunological effects on
polymerized flagellin. Furthermore, at low concentrations yl and IgM
enhanced the antibody response and suppressed the delayed hypersensitivity
induced by flagellin, whereas such an effect was not observed with poly-
merized flagellin or sheep erythrocytes. This result is consistent with earlier
studies which demonstrated that small amounts of passive antibody frequent-
ly enhanced the antibody response to soluble antigens but rarely to par-
ticulate antigens (Uhr & Moller 1968). One exception to this rule is the
 46 C. R. PARISH

TABLE I
The effect of different classes of passive antibody on the humoral and
cell-mediated immunity induced by monomeric and polymerized flagellin

Flagellin Polymerizeti flagellin

Passive Antibody
anti-flagdlin concentration
antibody Delayed-type Antibody Delayed-type Antibody
bypersensitivity response hypersensitivity response
Whole High
serum Low I + 0 0

High
Low 0 0 0 0

High 0
Low 0 0

y2 macrophage High ± -
absorbed Low 0 0

IgM
High ± 4-
Low 0 0

± = weak enhancement, + = enhancwnent, ++ = strong enhaocement.

0 = no effect, - = suppression, — = strong suppression.
(Adapted from Liew & Parish i972b)

report that IgM can sometimes enhance the primary antibody response to
sheep erythrocytes (Moller & Wigzell 1965, Henry & Jerne 1968).
Although regulaticm of the immune response by low concentrations of
passive antibody is certainly a complex phenomenon, several important facts
can be clearly discerned (Table I, and Liew & Parish 1972b,c). Firstly,
macrophage cytophilic antibody plays an important role in determining the
immunoregulatory properties of any antibody preparation, an observation
which will be discussed more fully in the following section. Secondly, low
concentrations of passive antibody tend to enhance the antibody response
to soluble antigens at the expense of delayed hypersensitivity, i.e., a positive
feedback was produced. This is in direct contrast to the negative feedback
observed with high concentrations of antibody and again suggests that serum
antibody modulates the immune response in such a way that both humoral
and cell-mediated immunity are expressed. Such a regulatory system does
not appear to apply for repeating determinant antigens, e.g., polymerized
flagellin and sheep erythrocytes. The possible cellular basis of these phenom-
ena will be discussed later in this review.
 HUMORAL AND CELLULAR IMMUNITY 47

To interpret meaningfully the effect of antibody concentration on humoral

and cell-mediated immunity, some estimate of the relative ratio of antigen
to antibody should be attempted. However, it should be remembered that
since flagellar antigens are much more rapidly eliminated from the recipient
animal than passive antibody (Shellam & Nossal 1971, Sinclair 1969), the
actual antigen to antibody ratio in vivo may be lower than that calculated
in vitro. Bearing this factor in mind, it was calculated that to induce en-
hanced cell-mediated immunity and suppressed antibody synthesis with
high concentrations of passive antibody, an approximately equimolar ratio
between antigen (flagellin) and antibody was required. In contrast, it was
calculated that ^1 antibody could enhance the antibody response to flagellin
with an antigen/antibody ratio as low as 2500/1, although slightly higher
antibody concentrations were usually needed to suppress delayed-type hyper-
sensitivity. Such a calculation could not be obtained with sheep erythrocytes
due to their complex antigenic nature.

B. Cytophilic Antibody
There have been several suggestions in the literature that macrophage
cytophilic antibody may play an important role in the suppression of anti-
body formation by passively administered antibody. Probably the most direct
evidence that macrophage cytophilic antibody may play an immunoregula-
tory role was published by Ivanyi (1970), who demonstrated that a passive
antiserum lost its immunosuppressive activity after removal of macrophage
cytophilic antibody by repeated absorption with spleen cells- Furthermore,
Sinclair et al. (1970) found that the removal of the Fc fragment from anti-
body drastically reduced its inhibitory effect, although others have not ob-
served such a reduction in inhibitory activity (Tao & Uhr 1966, Cerottini
et al. 1969). Less direct evidence is supplied by several workers who
observed that pre-exposure of spleen cells to antibody can markedly suppress
the subsequent antibody response to sheep erythrocytes (Rowley & Fitch
1964, Pierce 1969), although there have been reports that pretreatment of
lymphocytes with antibody failed to impair subsequent antibody responsive-
ness (Moller 1964, Uhr & Moller 1968). This apparent inconsistency in the
literature can probably be explained by the fact that the immunoregulatory
properties of macrophage cytophilic antibody are highly dependent upon
the type of antigen used (Liew & Parish 1972b,c). FOT example, macrophage
cytophilic antibody greatly influences the humoral and cellular immune
responses to flagellin and sheep erythrocytes but has little or no effect on
the response to polymerized flagellin (Liew & Parish 1972b).
Two lines of evidence have been obtained in this laboratory which suggest
48 C. R. PARISH

TABLE II
The immunogenicity of monomeric and polymerized flagellin bound
to macrophages via cytophilic antibody

Preparation Delayed Primary Secondary

Antigen
of antigen hypersensitivity antibody antibody

M - C A b - Ag
flagellin ±
POL •+ +

flagellin
M - C A b + Ag POL
flagellin
M-Ag POL
flagellin
M + Ag
POL

M = Peritoneal exudate cells ± = trace

CAb = Cytophilic antibody + — moderate
Ag = antigen ++ =1 high
POL = polymerized flagellin +++ = very high
(Adapted from Liew & Parish 1972c)

that cytophilic antibody for macrophages frequently can regulate humoral

and cell-mediated immunity. Firstly, it was found that yl and y2 antibodies
markedly differed in their effects on the immune response to flageUin and
sheep erythrocytes. The y2 fraction was rich in macrophage cytophilic anti-
body, whereas the yl fraction lacked this activity. However, absorption of
the cytophilic antibody from the y2 fraction with peritoneal macrophages
converted this antibody to a preparation which had immunological effects
approaching that of yl antibody (Table I, and Liew & Parish 1972b). Con-
versely, absorption of the j-l antibody with macrophages had no effect on
its immunolo^cal properties. Secondly, more direct evidence was provided
by the observation that the immunological behaviour of flagellin, poly-
merized flagellin and sheep erythrocytes was markedly altered when they
were injected bound to macrophages via cytophilic antibody (Liew & Parish
1972c).
Some of the immunological properties of macrophage bound antigen are
summarized in Table II. It can be seen that, as with circulating antibody,
the immunological effects were highly dependent upon the antigen injected.
Flagellin (and sheep erythrocytes) bound to macrophages either in the
presence or absence of cytophilic antibody induced higher levels of delayed
hypersensitivity than did free antigen. However, this cell-bound antigen was
less effective than free antigen at inducing a primary antibody response.
 HUMORAL AND CELLULAR IMMUNITY 49

On the other hand, polymerized flagellin bound to macrophages via cyto-

philic antibody failed to induce delayed hypersensitivity but induced antibody
formation more effectively than did free antigen. Similar results were ob-
tained when antigen was either complexed to macrophages with cytophilic
antibody or injected in the presence of macrophages sensitized with
cytophilic antibody. Finally, antigen bound non-specific ally to macrophages
behaved almost the same as free antigen. Thus, it appears that macrophage
cytophilic antibody enhances the cell-mediated immunity induced by soluble
(flagellin) and cellular (sheep erythrocyte) antigens at the expense of anti-
body responsiveness, whereas with filamentous antigens (polymerized flagel-
lin) it augments antibody synthesis. Undoubtedly this type of antibody
represents an important homeostatic mechanism in vivo as antigen bound
to macrophages via cytophilic antibody is not phagocytosed but remains
surface bound for long periods and can be immunologically active in
nanogram quantities (Liew & Parish 1972c).
This section has concentrated on the immunological properties of macro-
phage cytophilic antibody, but recently it has been demonstrated that anti-
body can be cytophilically bound to lymphocytes by either Fc (Basten et cd.
1972a,b) or complement (Bianco et ai 1970) receptors on the lymphocyte
membrane. The immunological significance of this antibody is not known,
although it has been proposed by Dukor et al. (1970) that the complement
receptor lymphocytes may transport antigen-antibody complexes to lymphoid
follicles, this antigen then more effectively triggering antibody formation.
The immunological significance of these cytophilic antibodies is currently
under investigation in this laboratory.

C. Antigen Degradation by Macrophages

Evidence has been steadily accumulating over recent years that small
molecules which possibly contain only single antigenic determinants may
be better inducers of delayed reactivity than are multideterminant molecules.
For example, as the molecular size and complexity of many artificial antigens
is expanded, they become poorer inducers of delayed-type hypersensitivity
but are more effective provokers of antibody formation (Jones & Leskowitz
1965, Stupp et ai. 1966). Similarly, the degradation of antigens into small
fragments has frequently resulted in preparations which induce good delayed
reactivity but poor antibody synthesis (Salvin & Liauw 1967, Lennon et ai.
1970, Pearson & Raffel 1971. Ichiki & Parish 1972b). These findings
suggest that macrophages, because of their capacity to degrade antigens
into small fragments, may play an important role in regulating humoral
and cell-mediated immunity in vivo. Evidence supporting this concept was

Transplant. Rev. (1972), Vol. 13 4

50 C. R. PARISH

TABLE III
A comparison of the humoral and cell-mediated immunity induced by
the peptic and cyanogen bromide fragments of flagellin

Induction
Primary Delayed-type
Molecular of
Antigen antibody byper-
weight antibody
response memorv sensitivity

Flagellin 40,000
Fragment A 18,000
(CNBr digest)
Fragments BCD 4,500-12,000
(CNBr digest)
High mol. wt. 13,000-15,000
peptic peptides
Low mol. wt. < 4,000
peptic peptides
Degraded high
moi. wt. peptic < 4,000
peptides

Immune responses tested against undegraded fiagellin.

(Adapted from Ichiki & Parish 1972b)

recently published by Pearson & Raffel (1971), who reported that sheep
erythrocytes which had been digested by macrophages lost the capacity to
induce antibodies but gained the ability to invoke delayed hypersensitivity.
Prelitninary studies have also indicated that macrophage degraded flagellin
may be a potent inducer of delayed hypersensitivity (M. Vennin^ un-
published).
A summary of the immunological properties of degraded flagellin is
presented in Table III (Ichiki & Parish 1972a,b). It can be seen that both
cyanogen bromide and peptic digestion of flagellin reduced the capacity of
the antigen to induce antibody formation or antibody memory, but enhanced
the ability of the molecule to induce flageUin-specific delayed-type hyper-
sensitivity. The hypersensitivity inducing peptides were found to be derived
from the region of the molecule which carried all of the antibody-forming
determinants, although whether the antibodies and delayed-type hyper-
sensitivity are directed against similar or different antigenic groupings on
flagellin is not certain at this stage. It should be emphasized that fragment
A carries all of the antigenic specificities of flagellin (Parish et al 1969)
 HUMORAL AND CELLULAR IMMUNITY 51

and therefore the more effective induction of delayed hypersensitivity by

this fragment cannot be due to it carrying a reduced number of antigenic
determinants. However, the fragment A determinants do have a reduced
affinity for anti-flagellin antibodies (Parish 1971c) and are therefore prob-
ably behaving like the antigenic groupings of the acetoacetylated flagellins.
There is now ample evidence that macrophages also play an essential role
in the induction of antibody responses to certain antigens. This has been
demonstrated clearly with the in vitro antibody response to sheep erythro-
cytes (Mosier 1967, Hartmann et al. 1970, Shortman et al. 1970, Shortman
& Palmer 1971). However, in marked contrast to the results with sheep
red blood cells, macrophage-free lymphocytes gave a full immune response
to polymerized flagellin (Shortman et ai. 1970, Shortman & Palmer 1971).
Recently Shortman & Palmer (1971) presented evidence which suggested
that macrophages merely degraded sheep erythrocytes into cellular fragments
which more effectively interacted with lymphocytes. Preliminary studies in
this laboratory have suggested, however, that although blockading of macro-
phages with colloidal carbon significantly reduced the antibody response
to sheep erythrocytes, there was a concomitant enhancement in the delayed-
type hypersensitivity to this antigen (Parish, unpublished).
Thus it appears that macrophages play an important immunoregulatory
role in vivo by either degrading cellular antigens into large fragments which
more effectively trigger antibody synthesis rather than cell-mediated im-
munity, or by cleaving multi-determinant antigens into smaller fragments
which preferentially provoke delayed hypersensitivity. The fact that circula-
ting antibody, via opsonisation, can enhance the breakdown of antigen by
macrophages presents another means by which antibody can indirectly
feedback on its own synthesis and divert the immune response into cell-
mediated immunity. The immunological role of macrophages in vivo may
not be as subtle as (a) supplying information RNA or RNA-antigen com-
plexes to antibody forming cell precursors (B cells) (Fishman 1969) or (b)
triggering B cells with antigen concentrated on its surface by T cell derived
cytophilic antibody (Miller et al. 1971).

III. THEORETICAL IMPLICATIONS

A. Does Immunological Unresponsiveness Exist?

One of the fundamental questions of immunology concerns the mechanism
by which an organism can immunologically reject foreign substances and
yet remain tolerant to self antigens. The most widely accepted explanation
of this phenomenon is the concept that when a clone of cells arises that is
52 C. R. PARISH

capable of recognizing and reacting against self antigens, that clone is elimi-
nated (Bumet 1959, Wilson & Billingham 1967). There is mounting
evidence, however, that tolerance does not always represent a completely
unresponsive state. Thus, situations have been described in which individuals
who were apparently 'tolerant' in vivo, actually possessed immune cells
capable of destroying the tolerated tissue in vitro, the activity of these
immune cells appearing to be blocked in vivo by serum factors (Hellstrom
& Hellstrom 1970, Voisin 1971, Wegmann et al. 1971). Crowle & Hu
(1969) have proposed that such 'tolerance' may be mediated by a 'hyper-
sensitization-inhibiting' or 'contrasensitizing' antibody in the serum.
Experimental data from this laboratory is also inconsistent with the view
that immunological tolerance represents a specific elimination of immujio-
logically reactive cells- The apparent inverse relationship between humoral
and cell-mediated immunity suggests that immunological tolerance frequently
represents a diversion of the immune response into either humoral or
cellular immunity rather than complete immunological unresponsiveness
(e.g., Figure 4). However, such an inverse relationship was only found to
apply when adult tolerance was investigated, neonatal tolerance being ex-
pressed at the level of both humoral and cell-mediated immunity and thw'e-
fore representing a completely unresponsive state (Parish 1971c, Parish &
Liew 1972). Thus it appears that immunocytes undergo a period during their
differentiation when tolerance to 'self antigens' can be induced, but once
these cells reach maturity a completely unresponsive state is unattainable.
It should be emphasized, however, that neonatal tolerance appears to be
readily abrogated by allogeneic lymphocytes (McCuUagh 1970, Voisin 1971,
Hellstrom et al. 1971, Wegmann et al. 1971), a finding which suggests
that tolerance is a specific repression, rather than an elimination, of im-
munologically reactive cells.

B. Are Helper Cells and Cellular Immune Cells Identical?

From a large body of evidence the conclusion is inescapable that the effector
cell in cell-mediated immune reactions is thymus-derived or thymus-de-
pendent (Bloom 1971). There is also ample evidence that the 'helper' cells
involved in the cooperative initiation of the antibody response are of thymus
origin (Rajewsky et al. 1969, Raff 1970, Mitchison 1971b,c, Miller et al.
1971). However, it is still not known whether the thymus-depcndent lympho-
cyte responsible for cell-mediated immunity and the helper cell in antibody
formation are functionally the same cell. There is some evidence, however,
for the following similarities between these two T-ccll functions:- (a) Both
cell-mediated immunity (Salvin 1958) and helper cell function (Cunningham
 HUMORAL AND CELLULAR IMMUNITY 53

& Ssrcarz 1971, Fidler et al. 1972) are initiated by lower doses of antigen
than antibody synthesis, (b) Helper and cellular immune cells mature
rapidly, reaching peak levels 4-6 days after immunization (Cunningham &
Ssrcarz 1971, Sell & Weigle 1959, Bloom 1971, Parish 1972b). (c) Both
T-cell functions exhibit similar levels of radiation resistance (Bloom 1971).
(d) The induction of both delayed-type hypersensitivity (Uhr & Moller 1968,
Liew & Parish 1972a) and helper function (Kappler et al. 1971) is much
less easily suppressed by passively administered antibody than is antibody
production, (e) Helper cells (Boak et al. 1971) and cellular immune cells
(Cooper 1972) have a similar anatomical distribution.
Recent studies in this laboratory may have shed some light on this
problem (Parish 1972b). Acetoacetylated flageUin can induce antibody
tolerance and delayed-type hypersensitivity in both low responder and high
responder rat strains. However, the antibody tolerance was only short lived
in the high responder strain (approx. 48 hr) but persisted for long periods
in the low responder animals ( > 28 days). The ability of the high responders
to recover from tolerance appeared to coincide with the development of
delayed-type hypersensitivity, but with the advent of antibody formation
delayed responsiveness declined. These results suggest that some T cells
associated with delayed-type hypersensitivity can 'help' antibody formation,
but following this participation can no longer express their hypersensitivity
properties.
On the other hand, when a classic hapten-carrier system was studied,
data was obtained which suggested that helper cells and cellular immune
cells are separate cell lineages (Table IV). In these experiments rats were
primed with different form^ of the flagellin carrier and helper cell function
then determined by challenging with DNP-flagellin. The experimental results
are summarized in Table IV. It was found that all three forms of flagellin
could induce substantial help; in fact, in the absence of carrier priming no
anti-DNP antibodies were detected 7 days after DNP-flagellin challenge.
However, acetoacetyl-flagellin, the most effective inducer of delayed hyper-
sensitivity to flagellin, was the poorest inducer of help, whereas polymerized
flagellin, a very poor inducer of delayed hypersensitivity, induced the
strongest help. These differences were particularly significant when lightly
substituted DNP-flagellins were used. A similar result has been obtained
with the in vitro antibody response to NIP-sheep erythrocytes (I. Trow-
bridge, personal communication). Periodate oxidized-acetoacetylated sheep
erythrocytes induce very good delayed-type hypersensitivity to sheep red
cells (Parish 1972a) but were much poorer inducers of helper cells for NIP
than were the unmodified sheep red blood cells which induce no detectable
delayed responsiveness. Thus it appears that delayed-type hypersensitivity
54 C. R. PARISH

TABLE IV
Lack of a relationship between cell-mediated immunity
to the carrier and helper function

Challenging
Priming Delayed-type antigen Anti-DNP
antigen hyperssnsitivity (1 ^g in saline) antibody*
(1 fxg in FCA) to flagellin (day 7)
(day 28)

Acetoacetyl- 5.9 ± 0.3 DNPs flagetlm 6.3 ± 0.3

flagellin DNPB flagellin 7.0 ± 0.4
(16.8 acetoacetyl DNP^1 flagellin 6.1 ± 0.5
groups/mole,
Krel = 6.8 x lO'

Flagellin 2.7 ± 0.1 DNP. flagellin 7.7 ± 0.3

D N P B flagellin 8.0 ± 0.2
DNP.^ 1 flagellin 7.8 ± 0.2

Polymerized 0.6 ± 0.2 DNP3 flagellin 9.2 ± 0.2

flagellin DNPe flagellin 10.6 ± 0.4
DNP« flagellin 7.7 ± 0.5

* In the absence of carrier priming no anti-DNP antibody was detected 7 days after
DNP-flagellin challenge.
Antibody titres expressed as logs, tube 1 = 1/10 dilution.
Hypersensitivity measurements represent 24 hr footpad swellings (1/lOth mm).
Each value represents the arithmetic mean of 7-8 rats ± standard error of the mean.
Krei = the antigenic activity of the antigen compared with unmodified flagellin.
(Parish 1971c)

to the carrier is not related to helper function in the anti-hapten antibody

response. In contrast, evidence has already been presented which suggests
that T cells associated with delayed hypersensitivity to the carrier can help
the anti-carrier antibody response. Tliis apparent paradox can be resolved
if a modification of the 'local environment' hypothesis is considered (Levine
1965).
It is well established that anti-hapten antibodies show a significant, al-
though limited, degree of specificity for structures on the carrier molecule
surrounding the haptenic grouping (Benacerraf et al. 1970). It has been
estimated that with anti-DNP antibodies the carrier contributes a binding
affinity of the order of 1000 calories/mole (Benacerraf et al- 1970). From
my observation that delayed-type hypersensitivity can be induced by flagellin
preparations which have drastically reduced affinities for anti-flagellin anti-
bodies is it not possible that the carrier could directly stimulate hapten-
carrier specific T cells? Under these circumstances it would be predicted that
 HUMORAL AND CELLULAR IMMUNITY 55

many of the helper cells for the anti-hapten response would be hapten
specific. Furthermore, due to the proposed low affinity of flagellin for
hapten-carrier specific T cells, the multideterminant polymerized flagellin
would be the most efficient stimulator of these cells. Chemical modification
would, if anything, reduce the capacity of the carrier to trigger such cells.
In contrast, delayed-hypersensitivity to the carrier would naturally correlate
with helper cells for the anti-carrier antibody response. Obviously from these
arguments I am proposing that the same T cell mediates delayed-type
hypersensitivity and helper activity. This may be a simplistic view when one
considers the functional heterogeneity of B cells, but it has advantages in
explaining homeostasis between humoral and cell-mediated immunity, a fact
which will be expanded upon shortly.
One additional point should be emphasized at this stage. Although the
carrier contribution to haptenic determinants has been widely recognized,
this contribution frequently has been dismissed as being too small to explain
the carrier specificity of the secondary antibody response to hapten-protein
conjugates (Benacerraf et al. 1970, Mitchison 1971a). It is noteworthy,
however, that reducing the affinity of flagellin for anti-flagellin antibodies
by only 1000 calories/mole (Parish 1971b) completely destroyed the ability
of the molecule to trigger both a primary (Parish 1971c) and secondary
(Parish 1972b) antibody response. A value of 1000 calories/mole has also
been estimated as the contribution of the carrier to the binding affinity of
anti-DNP antibodies (Benacerraf et al. 1970). When considering hetero-
logous carriers, due to steric hindrance, the influence of the carrier on
hapten binding could be much more pronounced (Benacerraf et al. 1970).

IV. A THEORETICAL INTERPRETATION

At this Stage a hypothesis will be outlined which will attempt to explain the
experimental data presented in this review. Before embarking on the hypo-
thesis, the following points should be noted:
(a) Only an adult (mature) immune system will be considered. As mentioned
earlier in this review, tolerance in neonatal animals appears to differ funda-
mentally from adult tolerance.
(b) It is assumed that B and T cells bear on their surfaces receptors which
reflect the specificity of the immune response which they will ultimately
express.
(c) It is proposed that a 'threshold energy of binding' of antigen with the
cell receptors must be achieved before a cell is activated. Thus, the activation
of cells would be an all or none phenomenon.
56 C. R. PARISH

(d) It is assumed that cell-cell interaction between B and T cells is necessary

for the induction of antibody formation- Of course, interaction between T
and B cells need not represent direct cell contact as indicated in Figure 6,
but may be mediated by antigen-specific products released by the T cells
(Feldmann & Basten 1972).
Figure 6 presents the hypothesis schematically. It is proposed that when
antigen binds to either T or B cells and achieves the appropriate threshold
energy of binding, the T cells are activated to proliferate and produce cell-
mediated immunity (and helper cells), whereas the B cells are rendered
'tolerant'. On the other hand, interaction between T and B cells with antigen
acting as a molecular bridge, results in antibody formation and cellular im-
munity tolerance. Thus, T cells can carry out only one function at a time;
either collaboration with a B cell to induce an antibody response or prolifera-
tion to produce cell-mediated immunity. Presumably the cellular immune
progeny are still capable of collaborating with B cells (see earlier).
With this hypothesis it is easy to envisage an inverse relationship between
humoral and cell-mediated immunity. Presentation of antigen in a form
which would favour cell-cell interaction would preferentially induce anti-
body formation, whereas the separate interaction of antigen with T and B
cells would favour cell-mediated immunity and antibody tolerance. Bearing
this concept in mind, the immunological behaviour of a number of antigenic
systems presented in this review can be described as follow^:-

Pigure 6. A schematic model of the immune response. For explanation, see text.
(Reproduced from Parish 1971c).
 HUMORAL AND CELLULAR IMMUNITY 57

(a) Monodeterminant antigens or antigens with a reduced number of deter-

minants tend to induce delayed hypersensitivity (and antibody tolerance) due
to their inability to mediate cell-cell interaction.
(b) Conversely, multideterminant antigens (e.g., polymerized flagellin) favour
antibody formation rather than cell-mediated immunity.
(c) Low doses of antigen preferentially induce delayed-type hypersensitivity
and antibody tolerance (i.e., low zone tolerance) as the probability of small
quantities of antigen inducing cell-cell interaction is low, whereas this amount
of antigen can probably attain the 'threshold energy of binding' required to
activate cells.
(d) Very high doses of antigen can produce enhanced cell-mediated immunity
and antibody tolerance (high zone tolerance), as at this dose level antigen
molecules tend to compete with one another for receptors on immuno-
competent cells, i.e., cell-cell interaction is inhibited.
(e) High concentrations of passively administered antibody can suppress
antibody formation and enhance delayed hypersensitivity by effectively
reducing the number of exposed antigenic determinants on antigens. Thus,
the probability of antigen-mediated cell-cell interaction is lowered and the
separate interaction of antigen with immunocompetent cells is favoured.
(0 Very low concentrations of passive antibody can aggregate soluble anti-
gens (e.g., flagellin) and present them in a form which more efficiently
induces cell-cell interaction (i.e., antibody formation). Such a phenomenon
obviously does not occur with large, repeating-determinant antigens,
(g) Antigens bound to cell surfaces, due to their separate presentation to T
and B cells, can induce delayed hypersensitivity rather than antibody forma-
tion. A similar phenomenon would be expected to occur whether the anti-
gens represented true surface structures or were bound via cytophilic anti-
bodies. Conversely, to induce antibody formation cellular antigens (e.g.,
sheep erythrocytes) must be degraded into small fragments (e.g., by macro-
phages) in order to mediate intimate contact between T and B cells,
(h) The affinity of antigen for the receptors on T and B cells is of crucial
importance in determining the extent of cell-cell interaction and, therefore,
the magnitude of the antibody response. If the affinity of an antigen for
cellular receptors is lowered (e.g., by chemical modification, CNBr digestion)
the ability of the antigen to induce antibody formation is rapidly destroyed,
whereas, if the modified antigen, can still attain the threshold energy of
binding required to activate cells, antibody tolerance and cell-mediated
immunity is induced.
This hypothesis also presents a new insight into the specificity of humoral
and cellular immune responses. It frequently has been proposed that the
antigenic determinants for humoral and cell-mediated immunity differ (Gell
58 C. R. PARISH

& Benacerraf 1961, Schlossraan & Levine 1967, Turk 1967, Schlossman
1972). This proposal has primarily been based on the observation that the
delayed-tyi>e hypersensitivity induced by a hapten-carrier complex is con-
jugate specific, whereas the antibodies produced are hapten specific- How-
ever, the data summarized in this review strongly suggests that, according to
the conditions of immunization, an antigenic determinant can have either
antibody or delayed-type hypersensitivity directed against it. This inter-
pretation implies that the receptors for antigen in both T and B cell popu-
lations have similar, if rot identical specificities but, as described above,
differ in their triggering mechanisms. Based on the hypothesis that antigen-
mediated T-B interaction is necessary to trigger antibody formation whereas
cell-mediated immunity is merely induced by antigen reaching a certain
threshold energy of binding with T cell receptors, it would be anticipated
that antibody formation (i.e., cell-cell interaction) would only be induced
against determinants with the highest binding affinities for cell receptors.
The obvious antibody forming determinants would therefore be the haptenic
groupings. Furthermore, any T cells with hapten specificity, via their inter-
action with B cells, would be prevented from proliferating to produce
hapten-specific cell-mediated immunity. In contrast, determinants with a
lower affinity of binding with cell receptors (i.e., some carrier determinants)
would stimulate delayed hypersensitivity and perhaps even antibody tol-
erance. Thus, the illusion would be created that T and B cells recognize
different antigenic groupings, whereas the difference lies in induction, not
in the specificity of recognition.
It should also be emphasized that the balance between humoral and cell-
mediated immunity can be exquisitely sensitive to changes in antigenicity.
For example, only a 275 calorie reduction in the affinity of flageUin for
anti-flagellin antibodies produced a 90-95 per cent fall in the antibody
response to flagellin (Parish 1972b). In contrast, destruction of the poly-
merizing sites of flagellin by chloramine-T oxidation had no effect on the
antigenicity or antibody-forming capacity of the molecule (Parish & Stanley
1972).
Assuming the lower affinity requirements for the induction of delayed
hypersensitivity, one would predict that many antigens which are closely
related structurally, although showing little or no cross-reacting antibodies,
should produce cellular immunity which strongly cross-reacts. In fact,
several different strains of flagellin, although producing weakly cross-reacting
antibodies (R. Langman, manuscript submitted), cross react strongly at the
cellular immunity level (Parish 1971b, Cooper 1972). Furthermore, it has
been observed that different species of red cells which do not cross react
at the antibody level provoke cross-reacting helper cells (Falkoff & Kettman
 HUMORAL AND CELLULAR IMMUNITY 59

1972, Hoffmann & Kappler 1972) and delayed-type hypersensitivity (Parish

1972a). A similar cross-reactivity has also been observed between serum
albumins (Gell & Benacerraf 1959). Antibody tolerance also should cross-
react in a similar manner if the proposition is correct that such tolerance
is induced merely by antigen which reaches a certain threshold energy of
binding with B cell receptors. Evidence consistent with this view is the
observation that the induction of neonatal tolerance with S- adelaide flagellin
produces partial antibody tolerance toa range of other, antigenically different,
flagellins (Austin & Nossal 1966). Similarly it has been reported that there
is no straightforward correlation between the specificity of antigen-antibody
interaction and that of cross-tolerance between a range of synthetic poly-
peptide antigens (Bauminger & Sela 1969).

V. PRACTICAL IMPLICATIONS

From the experimental data summarized in this review several practical

implications can be drawn. Perhaps the most exciting is the possibility of
'immunological engineering'. Numerous studies have demonstrated that cell-
mediated immunity is of paramount importance for the rejection of tumours
(Hellstrom & Hellstrom 1970) and for recovery from many viral (Blanden
1971), bacterial (Mackaness & Hill 1969, Turk & Bryceson 1971), protozoal
and fungal infections (Turk & Bryceson 1971). In contrast, serum antibody
is the most effective fonn of immunity for eliminating toxins and invading
organisms from the circulation (Payling Wright 1968). It now seems feasible
that, by the appropriate modification of antigens, vaccines can be obtained
which selectively provoke either cell-mediated immunity or antibody for-
mation.
The selective induction of ceil-mediated immunity by chemically modified
antigens appears to be the technique with the greatest potential. Preliminary
experiments in this laboratory already have indicated that immunization
with periodate oxidized-acetoacetylated Ehrlich ascites tumour cells may
render animals resistant to a subsequent, lethal dose, of tumour. Vaccination
with the unmodified tumour had little or no effect on survival.
The demonstration of an inverse relationship between humoral and cell-
mediated immunity may also explain the frequent clinical observation, par-
ticularly well demonstrated with leprosy, that there exists a spectrum of
immunity to many infections, immunity ranging from almost exclusively
humoral to predominantly cellular in nature (Turk & Bryceson 1971).
Finally, the observation that adult tolerance, rather than being complete
unresponsiveness, actually represents a deviation of the immune response into
60 C. R. PARISH

either humoral or cell-mediated immunity, places serious dooibts on the

possibility of obtaining true immunological tolerance to tissue grafts. Other
approaches, such as the selective stimulation of enhancing antibody or the
artificial removal of graft-specific immunocompetent cells, must be explored.

VI. SUMMARY

In this review an attempt has been made to summarize recent experimental

data which suggest that an inverse relationship exists between humoral and
cell-mediated immunity. Based on these studies, some of the factors which
regulate humoral and cell-mediated immunity in vivo have been discussed
and a hypothesis proposed to explain their mode of action. It is proposed
that presentation of antigen in a form which favours T-B cell interaction
preferentially induces antibody formation, whereas the separate interaction
of antigen with T and B cells favours cell-mediated immunity and antibody
tolerance. This simple hypothesis can readily explain such diverse phe-
nomena as high and low zone antibody tolerance, the preferential induction
of cell-mediated immunity by monodeterminant rather than multideterminant
antigens, antibody feedback, the immunogenicity of macrophage bound anti-
gen, the immunological behaviour of chemically modified antigens and the
apparent differences in the specificity of humoral and cell-mediated im-
munity. The relationship between helper cells and cellular immune cells
is reviewed and evidence is also presented which suggests that neonatal
and adult tolerance represent different immunological states. Finally, based
on the evidence provided, the possibility of 'immunological engineering' is
briefly discussed.

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