In contrast to the LT induced activation of caspase-1, which can take up to 60 min [115
], MKK cleavage is extremely rapid, occurring within 20 min of LT addition to cells in vitro
]. The MAPK cascades are critical in controlling T-cell activation, as these pathways are involved in the activation of T cell receptor (TCR) dependent nuclear factor of activated T cells (NFAT) family members. Along with activating transcription factor 2 (ATF2), the NFAT family members act synergistically with nuclear subunit activation protein 1 (AP-1) as transcription factors regulating the expression of inducible genes vital to the adaptive immune response. The regulation of NFAT, AFT2 and AP-1 activation is controlled at multiple levels by MAPK phosphorylation, the action of LT upon the MKKs thus blocks JNK, ERK and p38 mediated T cell proliferation [117
]. The NFAT transcription factors are also involved in the regulation of genes encoding IL-2 and the IL-2 Receptor (IL-2R), which are essential for the clonal expansion of antigen specific T cells. LT induced impairment of T cell proliferation is traditionally associated with the reduced production of the Th1 and Th2 cytokines IL-2, IFNγ, TNFα and IL-5 and the downregulation of the activation markers, CD69 and CD25 [117
]. The blockade of T cell activation being potent enough to inhibit the co-stimulation ex vivo
of LF exposed CD4+ T cells, by α-CD3 and α-CD28 antibodies [118
]. Due to its role in the perturbation of PKA dependent intracellular signalling pathways, EF also has an indirect effect upon NFAT transcriptional regulation, interfering with the calcium dependent signalling, but only when present at much higher concentrations than LF [117
The antagonistic effect of EF upon PKA strengthens the effects of LF on MAPK cascades, as EF is capable of both phosphorylating a negative regulatory residue of Raf, which lies upstream of the MAPK pathway, and phosphorylating a positive regulatory residue of Rap1 which inhibits Ras initiation of the MAPK cascade [120
]. The synergistic effect of LF and EF upon the MAPK pathways suppresses T cell chemotaxis in response to CXCL12 [120
]. This may be mediated through inhibiting the phosphorylation of both regulatory components such as paxillin and structural components such as actin, of the cell cytoskeleton [121
]. The elevation of intracellular cAMP by EF also appears to skew the differentiation of naïve CD4+ T cells towards a Th2 subset, inhibiting the TCR dependent activation of Akt1, a protein essential for the development of a Th1 subset, whilst enhancing the activation of the guanine nucleotide exchanger Vav1 and the stress kinase p38 which are involved in Th2 differentiation [123
]. A predominantly Th2 response has also been reported following stimulation of PA-specific T cells from AVA immunised rhesus macaques typified by increased levels of IL-4 and IL-2 but not IFNγ, IL-6, IL-1β or TNFα mRNA levels [18
]. Boyaka et al.
have also found that PA specific CD4+ T cells from mice immununised with rPA secrete low levels of Th1 associated cytokines, but increased levels of characteristic Th2 cytokines IL-4, IL-5 and IL-10 [124
]. It is important to note however, that both studies utilised adjuvants which may have had an impact upon the induction of distinct T cell subpopulations, as both the alum adjuvant in AVA vaccines and the cholera toxin adjuvant used by Boyaka et al.
have been implicated in biasing the CD4 T cell response towards a Th2 population [125
]. Along with the LF and EF effect upon DC cytokine release, which promotes preferential Th2 differentiation, this represents both direct and indirect consequences of anthrax toxins upon the lineage commitment of naïve CD4+ T cells.
The importance of an IFNγ CD4+ T cell response in mediating protection from anthrax infection in vivo
], in addition to the increased survival and B. anthracis
bactericidal activity of IFNγ activated macrophages [128
], and the disproportionate focus of LT upon suppressing IFNγ release by NK cells [129
], suggests that survival of B. anthracis
within the host depends in part, upon the suppression of a Th1 associated IFNγ release. Conversely this also indicates that the induction of an IFNγ producing memory T cell population should be an important goal of any future B. anthracis
Recent research has suggested a role for natural killer (NK) cells in the production of IFNγ following exposure to anthrax spores [130
], and natural killer T (NKT) cells in the generation of PA-specific, LT neutralizing antibodies by B cells [131
]. NKT-derived IL-4 and IFNγ influences the production of polyclonal PA-specific IgG1, which Devera et al.
found to be more protective in vivo
than polyclonal PA-specific IgG2b or IgG2c [132
]. However, it must be noted that Abboud et al.
found that PA-specific IgG2a isotypes were superior to both IgG2b and IgG1 in terms of in vivo
]. Devera et al.
acknowledge that due to the absence of IgG2a isotypes within their work, IgG1 may have assumed a greater importance than it might otherwise warrant, although the passive transfer of monoclonal antibodies in the model of Abboud et al.
could equally have led to a greater role for IgG2a than may yet be demonstrated within a natural anthrax infection.
The CD1d restricted type I invariant NKT (iNKT) cells provide B cell help, in addition to the production of the pro-inflammatory cytokines IFNγ, TNFα and GM-CSF. Surprisingly iNKT cells appear to express higher levels of the PA receptors TEM-8/ANTRX1 and CMG-2/ANTRX1 than other hematopoietic cells [134
]. As NKT cells represent less than 1% of the circulating T lymphocytes in humans (although this percentage varies substantially between individuals) this may denote a disproportionate focus upon this cell population by anthrax toxins. This preferential targeting of iNKT cells is supported by the downregulation of the activating receptor NKG2D on iNKT cells but not NK cells in the presence of LT [134
]. LT is capable of mediating a variety of effects upon iNKT cells through the cleavage and inactivation of MKK2 which impacts upon the phosphorylation of downstream ERK1 and 2 [134
]. Inhibition of the ERK pathway impairs CD1d mediated antigen presentation to iNKT cells, in addition to an observed reduction in endosomal trafficking of antigen loaded MHC class II molecules; this has major implications for the induction of both NKT and CD4+ Th1 adaptive immune responses to B. anthracis
The inhibitory effects of both LT and ET upon expression of the activation markers CD25 and CD69 and the secretion of the pro-inflamatory cytokines IL-2, TNFα, and IFNγ by human T cells has been described in vitro
]. Murine lymphocytes have also demonstrated impaired TCR mediated cell activation and CD4+ T cell production of the cytokines IL-3, IL-4, IL-5, IL-6, IL-10, IL-17, TNFα, IFNγ and GM-CSF following exposure to LT and ET [118
]. However, the cellular immunity identified within naturally infected humans indicates that, although in vitro
exposure to ET has been implicated in immune deviation towards both the Th2 and Th17 pathways [123
], the human immune response against LF encompasses a broad range of cytokines associated with Th1, Th2, Th9 and Th17 subsets, indicating little or no helper T cell polarization [114
]. This lack of skewing following repeated exposure to LF antigens echoes the work of the Peakman lab, who found that there was an equal balance of Th1 and Th2 responder cells in the recall immunity to anthrax [137
]. Martchencko et al.
] observed that anthrax toxin sensitivity, which varies between cell lines generated from different humans, appears to be strongly correlated with variation in the expression of the host cell surface receptor ANTXR2/CMG2. A single-nucleotide polymorphism (SNP), affecting the uptake of anthrax toxins, was identified in the protein coding region of ANTXR2/CMG2. Interestingly, the polymorphism, which is commonly found in African and European populations, decreased the cellular entry and subsequent lethality of anthrax toxin in murine cells, although this was not translated into a statistically significant difference between the in vitro
human cell populations, in terms of toxin sensitivity. It remains unclear, at present, what role the genetic diversity of the host plays in the course of a B. anthracis
infection, however two recent large scale studies have indicated differences in the generation of toxin specific antibodies between AVA vaccinees of different ethnicities. Crowe et al.
reported that African American individuals displayed lower titres of toxin neutralising antibodies following vaccination [36
], while Marano et al.
found that, during the immunisation schedule, anti-PA IgG levels were significantly lower in African Americans when compared to Americans of European descent (although this effect was not observed at a time point seven months post-vaccination) [139
]. As Ingram and Baillie observed, a range of genetic factors may govern this variation in response to anthrax derived proteins, including the expression of cellular receptors like ANTXR2/CMG2, polymorphisms in both cytokine and cytokine receptor genes, and variations in Toll like receptor (TLR) genes or HLA haplotype [140
Extensive allelic polymorphisms observed within the HLA region influence the repertoire of antigenic epitopes presented by MHC class II molecules to CD4 T cells; in order to fully elucidate the cellular immune response to anthrax it is therefore important to define these epitopes. Most vaccine strategies against anthrax have concentrated upon PA, however LF has been identified as a major target of T cell immunity in humans [49
]. As the amount of LF released by B. anthracis
is one-sixth of that of PA [141
], these findings indicate that LF may contain proportionally more epitopes which are of relevance in the T cell immune response. The elicited human T cell responses indicated that the key LF epitopes, LF574–593
, are concentrated in domain IV (). It is interesting to note that this reveals a very different pattern to that of the B cell epitope mapping performed by Nguyen et al.
, who found that the majority of structural B cell epitopes clustered in domains I and III of LF [41
The binding groove created by domains II, III and the catalytically active center of the LF toxin, domain IV, is responsible for holding the 16 amino acid long peptide which makes up the tail of the MAPK kinase family [63
], and it has been observed that mutations in the sequence coding for domain IV eliminates the peptidase activity of LF, abrogating its toxicity [142
]. The putative zinc binding site, which lies between the amino acid residues LF686
] was a feature of the response to LF674–693
; these represent important residues for zinc binding and catalysis as well as cytotoxicity, which are conserved across this class of metalloprotease [72
]. The immunodominant epitopes identified within domain IV therefore appear to comprise essential residues of LF which are critical for efficient catalytic activities and the execution of substrate cleavage.