Cytokines are soluble proteins or glycoproteins made by cells which affect the
behaviour of other cells. Regulatory mechanisms have evolved to keep cytokines under
tight control and these occur through various feedback loops to form a complex
cytokine network. T cells interact within this network and much of their development
and function are mediated by cytokines. In turn, the various T cell subsets (Thl, Th2,
T el, Tc2) are responsible for the expression and secretion of various cytokines as a
result of productive antigen stimulation.
The results in this chapter demonstrate the profound effect cytokines such as TNF-a, y-
IFN as well as IL4 have on lymphocytes and monocytes. These changes involved
molecules that are known to be intimately involved in antigen recognition and cell-cell
contact and support the association that exists between the pro-inflammatory “cytokine
storm” and subsequent development of GvHD. T cells were found to be most sensitive
to y-IFN and to a lesser extent, to TNF-a. IL4 had httle if any effect on T cells. The
changes seen on T cells were firstly, an increased density of class I allowing for more
MHC-peptides to be displayed on the cell surface and secondly, more T cells
expressing CD54, important both as an adhesion and costimulatory molecule in
productive antigen presentation. Monocytes displayed exquisite sensitivity with
exposure to cytokines and changes were seen in all molecules tested except for C D lla.
Like T cells, monocytes were most responsive to y-IFN but TN F-a and notably, IL4
also exerted potent effects. Besides changes in class I, class n and CD54, it would be
envisaged that the increased levels of CD80 and CD86 would enhance their capacity as
antigen presenting cells by providing adequate and essential co-stimulation. The only
a and only in monocytes. Surprisingly, it caused a downregulation of expression
although y-IFN did exhibit a trend for upregulation which was not statistically
significant. The natural hgands for CD49d include VC AM on APCs, MadCam-1 and
extracellular matrix proteins fibronectin and thrombospondin. It has been said that the
CD49d and VCAM system may be more important in the trafficking of leukocytes
across endothehal surfaces rather than in APC-T cell interactions (Schlegel 1997), (Itoh
et al. 2000), (Springer 1990).
B cells, hke monocytes are important antigen presenting cells. Unhke T cells and
monocytes, they were found to be most responsive to IL4 and to a lesser extent y-IFN.
Class I, CD54 and CD80 were the cell-surface molecules upregulated after exposure to
cytokines. Class II was already densely expressed on the cell surface at baseline and
cytokine exposure did not alter it further. The advantage of the flow cytometric method
employed in this study was that a variety of cell surface molecules on different
mononuclear cell subsets could be analysed simultaneously in one experiment.
The importance of adhesion molecules in GvHD was highlighted by studies which
demonstrated elevated levels of adhesion molecules in target tissues and a correlation
with the occurrence of GvHD: increased levels of VCAM-1 expression in the skin and
increased ICAM-1 (CD54) in the skin, gut, liver and endothehum (Norton et al 1992a),
(Norton et al. 1992b), (Norton et al. 1991b). Blockade of important adhesion molecule
interaction (CDlla-CD54, CD49d-VCAM) or costimulatory pathways (CD80:CD28)
using either monoclonal antibodies anti-LFA-1 and anti-VC AM antibodies or negative
signalhng molecules (CTLA-4) have also been shown to reduce the severity of GvHD
(Tanaka et al. 1995), (Schlegel et al. 1995), (Haming et al. 1991), (Blazar B.R. et al.
The results of this chapter have emphasised several important points regarding the
interaction of cytokines with lymphocytes and monocytes. For each cell surface
marker examined (MHC, costimulatory or adhesion), there was differential sensitivity
among the 3 cell types (monocytes, T cells and B cells) in their response to a particular
cytokine. As shown in Table 5.5, there was a similar baseline expression of Class I in
all 3 cell types but exposure to y-IFN highlighted the increased sensitivity of monocytes
compared to T or B cells. This differential sensitivity was even more obvious with
class II expression where all 3 cytokines, especially y-IFN induced a substantial
upregulation on monocytes while having no significant effect on T or B cells. In line
with this finding, it has previously been reported that Class II expression on human T
cell hnes is regulated by other factors other than y-IFN (Gerrard et al. 1988)
Conversely, for a particular adhesion molecule, each cell type showed different
sensitivities to different cytokines. For example, in CD54 expression, TN F-a was the
most potent stimulator of increased expression in monocytes whereas in T cells, it was
y-IFN and in B cells, IL4 that exerted the greatest effect.
For each cell surface marker, the density of expression and the proportion of positively
expressing cells were the two parameters measured. It was found that cytokines could
alter both: a greater percentage of cells expressing the adhesion molecule without
altering its density (CD54 in T cells), a higher density of expression in already positive
cells (Class I in T cells) or both (CD80 in monocytes). An important point to note
however, was that in this study, the antibody staining protocol used detected either the
presence or absence of the cell surface marker. It could be possible that cytokines
might affect a 3"^^ property of these adhesion or costimulatory molecule which is a
be operative for C D lla, expressed on all mononuclear cells and which somewhat
surprisingly, demonstrated no sensitivity to any of the cytokines tested in contrast to its
Hgand CD54. It is however well documented that C D lla exists in more than one
conformational form and it may be that cytokines induced a transformation from a low
to a high affinity state (Cabanas & Hogg 1993). Unfortunately, this could not be tested
in the antibody and assay system used.
Increasing the dose of cytokines in nearly all the experiments did not increase the effect
of the cytokine, that is, lOOOU/ml y-IFN was as effective as 4(XX)U/ml y-IFN. This was
not surprising as the effect of cytokines is often exerted within the local milieu of the
secreting cell. Hence systemic concentrations are less vital than the local presence of
these cytokines in influencing cellular contact or antigen presentation.
The effects seen with the cytokines were evident and maximal at 24 hours. The 48
hour time point confirmed that these effects were sustained up to 48 hours. It remained
possible however that certain changes induced might only appear later on and therefore
not be detected by this study. How sustained this cytokine induced changes were
beyond 48 hours was also not addressed. The initial changes seen during these time
points were of greatest interest to us as the allodepletion strategy was based on CD69
upregulation at 72-96 hours.
The cytokine cascade in GvHD highhght the intimate interactions between cytokines
and this complex network was illustrated by results in this chapter showing that y-IFN
and TNF-a together often exerted an additive (class I in T cells) or even synergistic
(CD54 in T cells) effect although in one instance (CD49d in monocytes), their actions
important clinically because in many inflammatory situations including GvHD, y-IFN
and TNF-a are produced and released in large quantities. Different T cell subsets
produce distinct cytokines with y-IFN secreted by Thl cells and IL4 by Th2 cells. The
more dramatic changes seen with y-IFN in this study lends support to the idea that acute
GvHD is associated with a dramatic skewing towards a predominantly Thl repertoire.
Besides having a central role in cell-mediated immunity, y-IFN exerts profound effects
on leukocyte-endothelium interactions and is involved in the activation of granulocytes,
macrophages and natural killer cells (Boehm et al. 1997). TN F-a is the prototype pro-
inflammatory cytokine and has been implicated as one of the prime effectors in
producing the deleterious effects seen during acute GvHD (Hill et al 1997b). The data
presented here suggest that it also influences the initiation of antigen recognition, singly
or more significantly, in association with y-IFN.
The interactions described here between cytokines and the changes in the MHC,
costimulatory and adhesion molecules seen on monocytes and lymphocytes has
provided the basis for understanding the mechanism of the modified MLC assay used
in the allodepletion strategy. It has also further elucidated the complex role that the
“cytokine storm” plays in the initiation of the GvH reaction including a vital role in
antigen and allogeneic recognition. In terms of tumour immunology and the GvL
response, it has been shown that cytokine treatment can lead to the induction of
costimulatory and adhesion molecules on leukaemia blasts. This then allowed for
productive anti-tumour T cell responses, confirming the role of cytokines in influencing