Immunity to Protozoa

Relative Role of B and T Cells in Immunity to Protozoa

Parasites with complex life cycles often stimulate both antibody- and cell-mediated defence
mechanisms, whose effectiveness depends on the particular parasite and the stage of infection.
Malaria, which is caused by different species of plasmodia, ranks amongst the most common and
important parasitic diseases. Infection of humans is initiated by the biteofan infected mosquito,
resulting in the injection of sporozoite stages. The sporozoite enters hepatocytes where asexual
reproduction (schizogony) takes place. Rupture of the infected cells releases
thousandsofmerozoites,which then penetratered blood cellsto initiate repeated erythrocytic
cycles. Thus, the different stages of the parasite occur inside cells that either express (e.g.
hepatocytes) or lack (e.g. erythrocytes) major histocompatibility complex (MHC) class I or class
II molecules. Since T cells are able to recognize parasite antigens only as processed peptides
presented by appropriate MHC molecules, extracellular formsofthe parasite, or parasite stages in
erythrocytes devoid of the presentation machinery, are controlled mainly by antibody dependent
acquired immune effector mechanisms. However, the immunological control is only partial:
complete elimination of plasmodia is not achieved.

Role of antibodies

In both humans and mice, passive transfer of antibodies from immune individuals to those
suffering from acute malaria results in quick and marked reduction of parasitaemia. In addition,
infection with Plasmodium berghei and P. yoelii cannot be controlled in mice from which B cells
have been removed by neonatal anti-m treatment (Grun and Weidanz, 1981). The elimination of
parasites is also impaired in mice with targeted gene deletions resulting in B-cell deficiency
(vander Heydeetal., 1994; von der Weid et al., 1996). While immunoglobulin (Ig) G2 aisessential
in the mouse model (Whiteetal.,1991), IgG 1 and IgG 3 (Grouxand Gysin, 1990) appearto be
most effective in humans. IgG1 and IgG 3 in humans and IgG 2 a in mice are cytophilic
immunoglobulin isotypes that are able to promote activation of monocytes and macrophages via
Fc receptors. The antibody-dependent killing of parasites in vitro is dependent on the presence of
either mouse neutrophils or human monocytes. One mechanism possibly involved in this
antibody activity is the induction of tumour necrosis factor (TNF) a secretion by monocytes,
leading to growth inhibition of intracellular parasites in neighbouring cells. Selective
agglutination of infected erythrocytes is consistently associated with reduced parasite density.
Opsonizing and agglutinating antibodies recognize a group of large proteins inserted in the red
cell membrane by mature asexual blood stage parasites. These proteins, which interact with a
variety of endothelial receptors such as CD36, E-selectin, thrombospondin, vascular cell
adhesion molecule 1 and intercellular adhesion molecule 1, play a critical role in the retention of
infected erythrocytes in the blood vessels and, thereby, protect the parasite from sequestrationin
the spleen.Thus, an important function of antimalaria antibodies may be the inhibition of
endothelial] blood cell interaction which ultimately promotes the elimination of infected
erythrocytes by phagocytes in the spleen.

Role of T cells

Two stages of the malaria parasite are truly intracellular: the sporozoites that infect the
hepatocytes and the asexual merozoite stage that resides in red blood cells. Infection with
plasmodia stimulates both CD41 and CD81 T cells expressing anaboragd T-cell receptor (TCR).
Whilemice genetically deficient forab TCR T cell sarevery susceptible to P. chabaudi chabaudi
infection and die rapidly after infection, there is no difference between gd TCR-deficient mice
and control mice (Langhorne et al., 1995; Sayles and Rakhmilevich, 1996). CD81 T cells
mediate killing of the liver stage of plasmodia, possibly by producing cytokines (interferon g
(IFN-g), TNF) which induce the production of nitric oxide by infected hepatocytes (Hoffman et
al., 1996). The central role of CD41 T cells in the protective immunity against the asexual blood
stages of experimental malaria has been shown by in vivo cell depletion analysis and by cell
transfer studies. Since transfer of purified CD41 T cells or CD41 T-cell lines to severe combined
immuno deficient or lethally irradiated mice clears the infection only in the presence of B cells,
T]B-cell interaction is thought to be required for the establishment of afully protective immune
respon setomalaria parasites. In plasmodia-infected mice both T helper (TH) 1-type and TH2-
type CD4 T cells are involved in protective immunity (Taylor-Robinson et al., 1993). However,
the relative contribution of these subsets changes during the course of infection: TH1 cells
predominate during thea cute phase, and TH2 cells are found primarily during later phases of
infection. The protective effect of transferred TH1 cells in mice infected with P. chabaudi
chabaudi can be blocked by inhibitors of inducible nitric oxide synthase (iNOS), whereas
resistance conferred by TH2 cells is not affected (Taylor-Robinsonetal., 1993). Eveninthe caseof
TH1 cells, there appear also to be nitric oxide-independent mechanisms of protection, as shown
in P. yoelii-infected mice (Amante and Good,1997). Protective TH2 cell clones specific for P.
chabaudi chabaudi drive a strong protective malaria-specific IgG 1 response in vivo (seeabove),
which is promoted by interleukin (IL) 4. Given the central protective role of CD41 T cells in
murine models of malaria, it isstillpuzzling that there is no major effect of the human
immunodeficiency virus pandemicon the incidenceor severity of human malariaas has been
observed for tuberculosis, toxoplasmosis and leishmaniasis. It has been speculated that
progression to acquired immune deficiency syndrome (AIDS) involves a preferential depletion of
TH1-type CD4 T cells and not TH2-type cells, and that the latter might be sufficient to maintain
a protective antibody-mediated immunity to malaria.

Protection Versus Susceptibility Mediated by T helper Type 1 Versus Type 2 Cells

T cells can be subdivided on the basis of the expression of the proteins CD4 and CD8 into the
CD41 and CD81 subsets which have helper functions for other cells of the immune systeman
dcytolytic functions, respectively. After description of these subsets, CD41 T cells with mutually
exclusive helper functions were identified: those that stimulate antibody production and others
that mediate delayed-type hypersensitivity. In 1986, the basis for these different functions was
discovered by the description of two types of T helper cells which differed by the pattern of
cytokines they produced. TH1 cells were defined as producers of IL-2, IFNg and TNFb, whereas
TH2 cells produced IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13. Later, it was shown that TH1 and
TH2 cells develop from identical precursor cells under different stimulatory conditions which
include the presence of IL-12 (for TH1 cells) or IL-4 (for TH2 cells). The definition of TH1 and
TH2 cells was originally derived from results obtained from long-term in vitro cultured T-cell
clones. The first model demonstrating that similar patterns of coproduced cytokines were indeed
detectable in vivo was experimental mouse leishmaniasis, which develops after subcutaneous
inoculation of promastigotes of the protozoan parasite species Leishmania major. This parasite
invades phagocytes (most notably macrophages) and replicates intracellularly in its amastigote
form. Only macrophages that

have been activated by T helper cells, especially by their product IFNg, are able to kill the
parasites by mechanisms that include the production of nitric oxide. Interestingly, this disease
may have dramatically different outcomes depending on the mouse strain infected. While
resistant mice (such as C57BL/6) developaself-healing local lesion and acquireimmunity to
reinfection, susceptible mice (e.g. BALB/c) suffer from systemic spreading of the parasites and
finally die from massive hepatosplenomegaly, immunosuppressionandthe formation of immune
complexes. In cell transfer experiments, it was shown that both the protective and the susceptible
phenotype could be transferred into syngeneic animals by purified CD41 T cells. (Liew et al.,
1982). Hence,theseCD41 Tcellstakenfromthedifferentmouse strains behave completely different.
Not surprisingly, the protective CD41 T cells were subsequently shown to produce the TH1
pattern of cytokines, including the macrophage-activating cytokine IFNg. Instead, CD41 T cells
from susceptible mice produced a TH2 pattern of cytokines,includingthemacrophage-
deactivatingproduct IL-10 (Heinzel et al., 1989). The reasons why such a differential appearance
of CD41 Tcellsoccursinsusceptibleandresistantmicehave not yet been elucidated and are a matter
of extensive investigation. It is known thatno single geneis responsible
forthephenotype.Rather,aclusterofpossiblysixdifferent
genesappearstobeinvolved(Demantetal.,1996).Whatis known, and what leads back to the basic
findings on the generation of TH1 and TH2 cells, is that BALB/c (but not C57BL/6) mice
produce IL-4 within hours after inoculation of the parasites. If this early IL-4 is neutralized, the
mice develop a resistant phenotype. The cellular source of the early IL-4 has been identified to
reside within a population ofT cells that carry a TCR madeup ofthe Va8 and Vb4 families
(Launois et al., 1997). These T cells react with a L. major protein termed Leishmania homologue
of receptorsforactivatedkinase(LACK),andtheirdepletion creates a resistant phenotype in
BALB/c mice (Julia et al., 1996). However, this cell subset does not definitely explain the
susceptibility of BALB/c mice. First, it is at present unclear whether or not C57BL/6 mice have a
similar T-cell subset and which cytokines the cells might produce. Second, it is possible that the
preponderance of IL-4 secretionfoundwithinthisVa8/b4T-cellsubsetinBALB/c mice may already
be the consequence of a different, more proximal, alteration which is the real reason for the
phenotype.Inturn,thisalterationmayhavetheimportant consequence to lead to a bias of the Va8/b4
T cells to produce IL-4.

Role of CD41 Versus CD81 T Cells in the Control of Parasitic Diseases

InfectionswithLeishmaniaspp.andToxoplasmagondiiare often cited as prototypic examples for

parasitic diseases controlled by different subpopulations of T lymphocytes. As detailed above,
the resolution of human and murine cutaneous leishmaniasis (elicited, for example, by L. major
or L. mexicana) is critically dependent on the development of CD41 type 1 T helper cells (TH1)
which release IFNg and activate macrophages for the killing of intracellular Leishmania
amastigotes. Conversely, a lack of TH1 cells and/or the expansion of a different type of CD41 T
cells (TH2) is found during the nonhealing course of L. major infection in certain inbred mouse
strains (BALB/c, DBA/2) or in humans with chronic progressive visceral
leishmaniasiswhichiscausedbyL.donovaniorL.infantum (Reiner and Locksley, 1995). In contrast
to the essential role of CD41 T cells for both protection and disease, CD81 T cells appear to be
dispensable for a protective immune response towards Leishmania. Depletion of CD81 T cells
(by antibodies or via deletion of the b-microglobulinor theCD8achaingene)doesnotprevent cure
of infection with L. major in genetically resistant mouse strains (Wang et al., 1993; Huber et al.,
1998). However, CD81 T cells are not without function in
murineandhumanleishmaniasis.TheyproduceIFNgand induce the production of nitric oxide,
mediate resistance against reinfection, and are cytotoxic for parasite-infected macrophages (Mu¨
ller et al., 1994; Brodskyn et al., 1997). Whether the latter function is beneficial to the host
organism or contributes to progressive tissue damage as seen in chronic mucocutaneous
leishmaniasis is currently unclear. Similar to Leishmania spp., T. gondii is also an intracellular
protozoan which undergoes stage conversion after infection of mammalian host organisms.
Unlike Leishmania, which is usually taken up by macrophages via classical and coiling
phagocytosis, T. gondii tachyzoites actively invade nearly all nucleated cells, including
professional phagocytes, fibroblasts, endothelial and epithelial
cells,andastrocytes(Mauel,1996).Duringtheacutephase
ofinfection,T.gondiitachyzoitesrapidlyproliferatewithin these cells. The replication process is
finally controlled by the immune system which leads to a chronic infection, in which the
parasites persist lifelong as slowly dividing bradyzoites in tissue cysts.
Inthepast,anumberofdifferentmousemodelswasused to analyse the relative contribution of CD41
and CD81 Tcellsduringthevariousphasesoftheimmuneresponseto T. gondii. Adoptive transfer
studies showed that CD81 T cells from immune mice are most potent in mediating resistance to
primary infection with virulent T. gondii in immunocompetent naive recipients. Likewise, after
vaccination of mice with an attenuated T. gondii strain, depletion of CD81 T cells partially
reduces resistance to infection with live virulent T. gondii, whereas anti-CD41 treatment does
not affect the vaccine-induced resistance. Total abrogation of resistance, however, is achieved by
combined treatment of the mice with anti-CD4, anti-CD8 and anti-IFNg before the challenge
infection. In vitro, CD81 TcellsproducelessIFNg(andnoIL-2)inresponse
toT.gondiiantigen,comparedwithCD41 Tcells,butcan
beinducedtosecreteIFNgwhenexposedto(CD41 Tcellderived) IL-2. Mice that are depleted of
CD41 T cells before vaccination (with an attenuatedT. gondii strain) or infection (with an
avirulent T. gondii strain) never develop resistance to infection with a virulent strain of the
parasite. Similarly, when mice are orally infected with a lethal inoculum of virulent T. gondii,
CD41 T cells are required for longlasting survival of the mice after treatment with antiparasitic
drugs (atovaquone or sulfadiazine); mice depleted of CD41 T cells before infection rapidly
succumb to reactivated toxoplasmosis as soon as the drug treatment is discontinued (Araujo,
1992). In mice chronically infected with an avirulent strain of T. gondii, the asymptomatic latent
infection is fully reactivated upon treatment with anti-IFNg or a combination of anti-CD4 plus
anti-CD8 antibodies, whereas application of either anti-CD4 or anti-CD8 alone fails to enhance
brain pathology or mortality. Reactivation of latent toxoplasmosis was also observed in a mouse
model of retrovirus-induced CD4 immunodeficiency resembling HI virus-induced AIDS in
humans. Some mice survived the reactivation, which was dependent on the presence of both
CD81 T lymphocytes and IFNg (Gazzinelli et al., 1992). Together, the summarized results point
to a major effector role of CD81 T cells in toxoplasmosis, whereas CD41 T cells seem primarily
to assume regulatory (helper) functions essential for the generation of CD81 effectors. Both
CD41 and CD81 T cells release IFNg, whichisthekeycytokinefortheinductionofantimicrobial
activityinawidevarietyofhostcells.Knowntoxoplasmostatic effector mechanisms are the
generation of reactive oxygen intermediates (by reduced nicotinamide]adenine dinucleotide
(NADPH) oxidase), the production of nitric
oxide(byiNOS)andthedegradationofthetryptophan(by the indolamine 2,3-dioxygenase), which is
an essential amino acid for T. gondii. Alternatively, CD81 (and perhaps also CD41) T cells
might also confer protection through lysis of T. gondii-infected target cells (Montoya
etal.,1996).Astargetcelllysisdoesnotleadtothedeathof the intracellular T. gondii, the question
arises as to how tachyzoites are cleared in vivo. Possible effector mechanisms that might act
against released tachyzoites in the extracellular space include anti-Toxoplasma antibodies plus
complement, macrophages, natural killer cells and a subpopulation of cytotoxic CD81 T cells,
which were reported to be directly toxoplasmacidal.

Pathological Consequences of the Immune Response: Autoimmunity (Trypanosoma cruzi)

Infection of humans with the protozoon Trypanosoma cruzi results in American trypanosomiasis
or Chagas disease. The acute phase of Chagas disease is usually not fatal, subsiding within
2]4months, and is followed by a chronic phase in which the parasites are controlled by the
immune response of the host. However, they are not fully eliminated as they are able to persist
lifelong in humans in scarce numbers. Symptomatic chronic Chagas disease develops in 20]30%
of individuals infected with T. cruzi within 10]20years of infection. The heart is most commonly
affected. A T lymphocyte-rich inflammatory mononuclear infiltrate is observed in the cardiac
tissue (Higuchi et al., 1997), often together with interstitial fibrosis and atrophy of myocardial
cells, resulting eventually in death by disturbances of the cardiac conducting system or
congestive heart failure. Some patients present with serious pathological enlargement of the
oesophagus and/orcolon(megaviscera)associatedwithdysphagiaand severe constipation.

Pathogenesis of Chagas disease

The pathogenesis of the chronic cardiac or intestinal lesions is not fully understood, but an
altered immune response has repeatedly been implicated, suggesting that cross-reacting
autoimmune antigens deviate the parasiteinduced immune response towards the affected organs
of the host.

Autoimmunity

Such an autoimmune response could be induced either by T. cruzi antigens that show molecular
mimicry to heart proteins or by heart proteins that are liberated from cardiac cells during the
course of the parasite-driven acute myocarditis. Several investigators have found crossreactive
antibodies in T. cruzi-infected patients, directed mainly towards ubiquitous and evolutionary
conserved molecules. Many of these autoantibodies have been shown to be of heterophilic nature
(Petry and Eisen, 1989). Characteristically, the lymphocytic infiltrates are associated with
destruction of myocardial muscle cells. This destruction could result from the local effects of
lymphocytes against T. cruzi antigens, host antigens, or both. Several T. cruzi antigens have been
identified that show molecular mimicry with host cellular components, including a ribosomal
protein termed phosphoprotein (PO) and the cardiovascular b1-adrenergic receptor which trigger
theinductionofautoantibodies,butonlyfewofthemseem to be associated solely with Chagas disease
(Ferrari et al., 1995). Of special interest is the immunodominant T. cruzi B13 antigen, which
induces antibodies against the cardiac myosin heavy chain and stimulates myosin heavy
chainspecific T cells present in all patients with chronic Chagas cardiomyopathy (Cunha-Neto et
al., 1996). The myosin heavy chain is the most abundant heart protein (50% of total protein by
weight) and is recognized in many conditions of heart-specific autoimmunity, such as
rheumatoid heart disease and murine post-Coxsackie B3 autoimmune cardiomyopathy. Most of
the evidence, however, suggests the participation of T cells rather than of antibodies in heart
destruction. An analysis of the phenotype of the T cells infiltrating affected heart tissue suggests
a 2 to 1 preponderance of CD81 cells over CD41 cells, many of which express granzyme A as a
marker for cytolytic capability. A direct relationship has been observed between the number of
infiltrating lymphocytes and the propensityoftheimmuneresponseagainstthelocalloadof parasites
as well as the start of the tissue-destructive lesions. This scenario suggests that the tissue
destruction of the heart tissue is the effect of an immune response triggered by parasite-specific
CD41 and CD81 T lymphocytes, which via cross-reactivity to host self antigens augment the
damaging process (Tarleton et al., 1996). It remains to be shown whether the myosin-specific T
cells are able to initiate and support the cardial inflammation, or whether these cells are just
passively recruited to the inflamed tissue.Macrophages seem to play, if at all, only a minor role
in cardiac lesions.
Experimental murine Chagas disease

In mice, T. cruzi reproducibly induces different mild cardiomyopathy but does not lead to
intestinal disease. CD41 T cells from chronically infected mice were shown to transfer the
capacity for heart damage, whichwas taken as evidence of a T cell-mediated autoimmune process
(Kierszenbaum, 1995).

Role of Antigenic Variation (African Trypanosomes)

Antigenic variation describes the ability of a microorganism to change the antigenic make-up of
its surface proteins, and there by to evade the deadly immune response of the mammalian host
and to cause a chronic infection. In the early twentieth century, it was observed that sleeping
sickness, which is caused by African trypanosomes, is associated with success ivewavesof
parasitaemia. It is now generally accepted that this wavelike mode is the result of the continuous
generation of trypanosome variants exhibiting a distinct antigenic identity. The trypanosomes are
destroyed by host antibodies. However, some trypanosomes have altered their antigenic surface
coat and are able to escape eradication. They start a subsequent wave of multiplication and
continue the infection. Antigenic variation can be detected by various immunological methods.
Binding of antibody to a given variant antigenic type (VAT) can be measured by agglutination,
complement fixation, immunofluorescence or neutralization of parasite infectivity.

Molecular basis of antigenic variation: surface glycoprotein coat

The fixation of antibodies to bloodstream trypanosomes indicates the presence of antigenic

material on the surface of the parasite. This can be shown by electron microscopy using labelled
antisera which reveal a dense surface coat. The coat covers the plasma membrane and consists
primarily of 5]10106 molecules of a single variable surface glycoprotein (VSG) (Cross, 1990).
Trypanosomes ofdifferentVATdifferwithrespecttotheirVSG.TheVSG has a molecular weight of
about 60kDa and comprises about 10% of the dry weight of the parasite. Its major biological
function is to provide trypanosomes with a physical shield, preventing potentially adverse host
molecules from interacting with the plasma membrane of the parasite. The VSG coat is essential
for survival of the parasites in the blood of the host, because it is antiphagocytic, prevents
complement-mediated lysis and provides thebasisforantigenicvariation,whichendowstheparasite
population with the ability to survive despite recognition and control by the host immune system
and subsequent immune elimination. Trypanosomes of a given VAT exhibit only a single VSG.
Epitopes of this VSG are the target of VAT-specific antibodies. Antigenic variation results in the
exhibition of a different VSG in the coat of a few parasites, thus producing a distinct VAT,
which is not affected by antibodies against the previous VAT, and thereby guarantees
continuation of the chronic infection. To allow for efficient and lasting parasite transmission, it is
of greater advantage for trypanosomes to reside in the host for long periods of time with a
somewhat fluctuating parasitaemia than to multiply in an unlimited fashion and rapidly to kill the
host, or to maintain low levels of infection that may be terminated by the immune response.
Structure of the variable surface glycoprotein coat

The molecular structure of the VSG is well characterized (Blum et al., 1993). VSG molecules are
homodimers that are inserted in the plasma membrane with a glycosylphosphatidylinositol
(GPI)anchor. The molecule is composed of about 400]500 amino acids and consists of a highly
variable N-terminal domain and a rather conserved C-terminal domain, both of which carry
carbohydrate side-chains. The variability of the N-terminal polypeptide chain, which is exposed
to the external surface of the parasite, is responsible for the distinct antigenic specificity of each
VSG. The carbohydrate side-chains, primarily those of the GPI anchor, represent structures that
contain cross-reacting epitopes. These epitopes are not exposed at the surface of the intact
parasite and therefore do not contribute to antibody-mediated parasite elimination.

Genetic organization of the variable surface glycoprotein

A single trypanosome can generate more than 100 antigenically different VSGs, but at a given
time expresses only one of the multiple (4 1000) VSG genes. The active VSG gene is expressed
from a telomeric expression site. VSG variation is encoded by the rather complex
trypanosomegenome.EachparasitecarriesthefullVSGrepertoire
ofitsstrain.T.bruceiparasitesswitchVSGsspontaneously at a rate of 1023 to 1027 per division
(Cross, 1990), independent of the host antibodies. Switching takes place by duplicate
transposition of a silent VSG gene into the active site or by activation of one of the other known
20 telomeric expression sites. The question why trypanosomes possess so many expression sites
is still not well understood. The sequence in which VSG genes are expressed during
sleepingsicknessisirregular,althoughnotfullyaccidental. This could be relevant for survival of the
parasite, as an orderlyuseofVSGgeneswouldputthehostintheposition to generate an antibody
response against a VSG early during the sequence of expression. The fortuitousness in the
production of VSG may be of importance for the evolution of a host]parasite system with many
donor sequences and several hosts. Antigenic variation is not a phenomenon unique to
trypanosomes. For example, the bacterium Borrelia hermsii causes relapsing fever in humans and
similar diseases in other mammals. Borrelia survive in the host by altering a surface protein,
termed the variable major protein.

Summary

Immunological control of the African trypanosome growth is mediated by antibody responses to

the VSG molecule. Owing to the variation of these surface coat
molecules,theparasitesarecapableofevadingdestruction
bytheimmunesystemofthevertebratehost.Thus,despite
theirrelevancetothecontroloftrypanosomegrowthVSGspecific antibodies do not generate
prolonged immune protection. Todate,itisnotknowntowhatextentotherpartsofthe immune system
contribute to control of the African trypanosomes and to defence of the host from adverse effects
of the parasites. Interestingly, as a consequence of infection with the parasites the immune
response of the host is greatly altered in that the production of specific antibodies and the
proliferation of T cells is significantly suppressed, whereas levels of CD51 B cells, known to
produce autoantibodies, are increased and macrophages are activated.