Commentary:

Priming of alloreactive T cells – where does ithappen?

Dela Golshayan and Robert Lechler

Department of Immunology, Imperial College, Hammersmith Hospital, London, UK

Host lymphocytes can recognize alloantigens directly on transplanted donor tissue or

indirectly after these antigens are processed and presented on host APC. Here, we outline the

features of alloresponses that distinguish them from responses to conventional antigens,

then we discuss various study systems that have examined where the priming of alloreactive

CD4+ and CD8+ T cells occurs. Finally, we discuss the implications of recent data which

suggest that direct responses originate in the graft itself whereas indirect responses are

initiated in the draining lymph nodes.

See accompanying article: http://dx.doi.org/10.1002/eji.200425309

Key words: Transplantation / Allorecognition / Direct and indirect pathways

1 Introduction

Whether adaptive immune responses to transplant

antigens are initiated within the organized structures of

secondary lymphoid organs or in the graft itself has long

been amatter of debate among transplant immunologists

[1, 2]. Several recent studies have attempted to

determine where alloreactive T cells are primed in

response to the placing of an allograft in vivo. The latest

of these studies is published in this issue of the European

Journal of Immunology [3] and adds an additional

dimension to this controversial question by the indepen-

dent analysis of direct and indirect alloresponses (Fig. 1).

2 Features that distinguish alloresponsesfrom responses to conventional antigens

Before considering any of the reported experiments in

detail, it is worth emphasizing that, in general, allor-

esponses are not qualitatively different from conventional

responses to protein antigens. Consequently it is

reasonable to assume that professional APC, namely

DC, are pre-eminent in the initiation of the immune

response against transplantation antigens. Naive T cells

respond to foreign antigens if these antigens are

presented in the context of appropriate co-stimulatory

signals and within secondary lymphoid organs because

these cells are inefficient at crossing vascular endothe-

lium to enter peripheral tissues [4]. This implies that, as

classically described for responses to microbial antigens

[5], the first meeting point between host naive Tcells and

transplant antigens, leading to the initiation of the

alloresponse, would be in the secondary lymphoid

organs rather than in the transplanted tissue itself.

There are however unique features of the immune

response in the transplant setting. First, the grafted

tissue contains its own APC that can trigger direct-

pathway alloresponses. In the early stages after trans-

plantation, tissue-resident antigen-loaded donor imma-

ture DC will migrate out of the graft via the blood and/or

lymph towards secondary lymphoid organs (graft-drain-

ing lymph nodes and spleen) where they will mature and

encounter recipient resting T cells. The trafficking and

maturation of donor DC is triggered by pro-inflammatory

signals produced as a result of tissue injury in the early

post-transplant period and is the cornerstone for the

initiation of effective adaptive immune responses [6]. A

second distinguishing feature of alloimmune responses is

that they involve two pathways, direct and indirect, as

illustrated in Fig. 1. We proposed over two decades ago

that, once donor bone-marrow-derived APC are de-

pleted, the immune response against an allograft is

maintained by indirect-pathway allospecific T cells [7].

The indirect pathway involves allogeneic MHCmolecules

that are shed from the graft being taken up, processed

and presented as peptides by recipient APC to the Tcells

circulating between secondary lymphoid organs.

[DOI 10.1002/eji.200425506]

Received 27/7/04Revised 31/8/04Accepted 1/9/04

Abbreviation: EC: Endothelial cell

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f 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de

When analyzing the location of Tcell priming and the type

of APC involved, there is a second issue to consider,

namely that the memory subset of circulating T cells

contributes to primary alloresponses. In adult humans,

where memory Tcells account for a bigger proportion of

the alloreactive T cell repertoire as compared with

experimental rodent models, approximately 50% of the

T cells responding in the ‘primary’ mixed-leukocyte

reaction are pre-existing memory cells. This reflects the

fact that direct alloresponses result from a cross-reaction

by Tcells with specificity for complexes of self-MHC with

peptide [8–10]. As memory T cells have different homing

patterns and have less-stringent activation requirements,

their involvement may help to explain the rapidity and

vigor of in vivo alloresponses.

However, this raises a further issue, namely the role of

graft parenchymal cells in priming and maintaining direct

pathway alloresponses. Activated vascular endothelial

cells (EC) express MHC class I and II as well as

expressing adhesion molecules. But these non-profes-

sional APC may be limited in their ability to effectively

activate Tcells, because of their limited expression of co-

stimulatory molecules and the brevity of the encounter

between T cells and EC. Indeed, alloantigen recognition

on graft parenchymal cells was shown in some in vitro

studies to induce allospecific T cell hyporesponsiveness

rather than activation [11, 12]. These findings were also

supported in vivo by the fall observed in the frequency of

T cells with direct anti-donor specificity in renal and

cardiac transplant patients [13]. Other studies have

challenged these conclusions by showing that EC could

induce the proliferation of allogeneic T cells and their

differentiation into effector Tcells, albeit during a five-day

in vitro co-culture [14, 15]. EC may also play a role in the

local recruitment and maturation of circulating host

monocytes into efficient APC [16]. It is true that, in the

setting of primarily vascularized allografts, EC of donor

origin offer a unique interface for donor–recipient cross-

talk during the lifetime of the organ and it is attractive to

hypothesize that they are implicated in acute and/or

chronic graft rejection, if not as initiators at least as

targets.

3 Systems used to study where alloreactiveT cells are primed

Turning to the various studies that have addressed the

anatomy of alloreactive T cell priming, there are several

important points that need to be borne in mind in the

interpretation of the reported results.

The first is that some studies have used skin allografts in

which the vascular endothelium is host, not donor,

derived [1, 17]. Other reports have used cardiac or renal

allografts, in which the vascular endothelium is of donor

origin [2, 18–21]. This has obvious implications for

alloantigen encounter by circulating Tcells. As previously

discussed, it remains a point of debate as to whether the

luminal surface of the vascular endothelium is a possible

site of T cell priming but, in theory at least, T cells

circulating in the bloodstream have the opportunity to

encounter alloantigens on the graft EC. If not activating

the T cells directly, by displaying graft alloantigens, EC

could promote the recruitment and selective transmigra-

tion of antigen-specific T cells at the site of inflammation

[22].

Second, the experimental systems used to address this

issue have varied enormously in terms of the frequency of

Fig. 1. Direct and indirect pathways of allorecognition. (A) In

the ‘direct pathway’, recipient T cells recognize intact

allogeneic MHC on donor APC. (B) In the ‘indirect pathway’,

allogeneic MHC molecules that are shed from the graft are

taken up and processed by the recipient APC to be

presented as peptides in the context of self-MHCmolecules.

3302 D. Golshayan and R. Lechler Eur. J. Immunol. 2004. 34: 3301–3304

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alloreactive T cells that have been employed. In their

study, Lakkis et al. showed that splenectomized aly/aly

recipients could not reject cardiac allografts, thus

demonstrating the role of secondary lymphoid organs

in initiating an alloresponse by naive T cells [20]. They

reached their conclusions using an adoptive transfer

model of polyclonal populations of T cells isolated from

naive or sensitized wild-type mice with a ‘physiological’

frequency of alloreactive T cells. In contrast, the study

published in this issue of the European Journal of

Immunology [3] used transgenic mice with a monoclonal

alloreactive TCR. The latter system has the potential to

yield somewhat misleading results, because of the highly

artefactual alloreactive T cell frequency. Although it may

be a rare event for naive T cells to traverse the vascular

endothelium, in a monoclonal T cell repertoire, any T cell

that accesses the graft is allospecific. This issue is also

relevant to the interpretation of the observations reported

by Kreisel et al. who investigated the possibility of

peripheral sensitization within a vascular allograft [21].

Using TCR-transgenic mice (BM3), they concluded that

mouse vascular endothelium could activate directly

allospecific CD8+ T cells in vitro and in vivo, leading to

the rejection of cardiac allografts.

This raises another important variable in the experiments

that have analyzed the question of the first encounter of

host T cells with transplant antigens using T cells from

TCR-transgenic mice, namely the activation require-

ments of the T cells. In some cases, such T cells have

rather aberrant properties; for example, the TCR genes

used to generate the BM3 (H-2k) transgenic mice with

specificity for H-2Kb were derived from a Tcell clone that

was CD8-independent in its activation [23]. This pre-

sumably reflects a particularly high TCR avidity. In the

experiments by Baratin et al. [3] the activation require-

ments of the TCR-transgenic T cells in response to their

two defined ligands were not studied in detail. Were the

responses to hemoglobin (Hb) + H-2Ek and to H2-Ep

equally dependant on mature DC, or was the direct

response to H-2Ep less stringent in its requirement for co-

stimulation?

The use of separated populations of CD4+ or CD8+ Tcells

for the study of the host immune alloresponses is also an

important variable. CD4+ T cells can initiate allograft

rejection through direct recognition of allogeneic MHC

class II antigens as well as indirect recognition of

allogeneic MHC peptides processed by self-APC. Both

pathways have been shown to help direct-pathway CD8+

T cells that eventually injure allogeneic MHC class I-

presenting target cells. Although less studied, CD8+ T

cells primed through the indirect pathway can also

mediate allograft rejection [24]. Furthermore, various

experimental models have demonstrated that CD4+ or

CD8+ T cells, alone, are sufficient to initiate rejection of

MHC I or II-mismatched allografts. Clearly, donor MHC I

antigenswill persist for the life of the graft. However, MHC

II antigens may be less abundant once donor passenger

leucocytes have migrated out of the graft, and died or

been eliminated. Hence, the direct-pathway CD4+ T cell

response is likely to be limited to the early post-transplant

period.

Finally, the relative contribution of the two major path-

ways of MHC alloantigen recognition, the direct and the

indirect pathways, needs to be addressed. This is the

attractive aspect of the study of Baratin et al. [3]. They

made intelligent use of amouse transgenic for a TCRwith

direct allospecificity for H-2Ep, and indirect specificity for

the mouse Hb b chain presented by H-2Ek. Thus by using

H-2p donors they could analyze the direct response,

whereas use of C57BL/6 mice transgenic for the

Hb(64–76) epitope allowed the study of the indirect

response following transplantation into an H-2k recipient.

The results suggest a clear distinction between the site of

priming of direct compared with indirect alloresponses.

The initiation of direct responses was largely visualized

within the skin graft, whereas indirect responses

appeared to originate in the draining lymph nodes, and

only later did activated Tcells appear within the graft. It is

not entirely clear why this distinction should be so abrupt.

If the direct response against donor alloantigens was

initiated by donor APC, such as Langerhans cells, there is

no obvious reason why infiltrating Tcells that had indirect

allospecificity would not have been activated within the

graft at the surface of infiltrating recipient DC.

4 Conclusion

In conclusion, it seems likely that the dominant pathway

for the initiation of both direct and indirect T cell

alloresponses involves DC of donor and recipient origin,

respectively. However, the data described in the paper by

Baratin et al. [3] highlight the fact that under some

circumstances priming can occur within the graft itself.

Future experiments should pursue these issues using

mice engineered to have much lower frequencies of

alloreactive T cells, using the adoptive transfer model

pioneered by Jenkins and colleagues, for example [25].

Similarly, the relevance of pre-existing memory T cells

and their contribution to the direct and indirect allor-

esponses should more carefully be examined.

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Correspondence: Robert Lechler, Department of Immunol-

ogy, Imperial College, Hammersmith Hospital, Du Cane

Road, London W12 ONN, UK

Fax: +44-208-3832788

e-mail: r.lechler@imperial.ac.uk

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