Despite the recognition that class II molecules present cytoplasmic Ags directly and indirectly, the principles underlying indirect presentation are poorly defined. Understanding this process is highly significant because TH
cells regulate antibody- and CTL-mediated adaptive immunity to pathogens, cancers, allografts and autoantigens. Such an understanding is especially important because many tumour and virus infected cells down regulate TAP gene expression to evade CTL-mediated immune surveillance. Furthermore, our findings will impact how we understand T cell responses in individuals that express TAP null and ERAAP variants (62
), especially those that inhibit or alter peptide processing within the ER.
We have shown here that indirect presentation of class II-restricted Ags requires CD8+ donor and recipient DCs. Within these cells, proteasomes, TAP and ERAAP—key components of the cytoplasmic Ag-processing pathway—regulate indirect presentation of class II-restricted Ags thereby impacting the magnitude of TH cell responses to cytoplasmic alloantigens (HY and H3b) and bacterial (L. monocytogenes) Ags. Because these effects were observed with two distinct models, we suggest that the impact of the CAP machinery on indirect presentation of cytoplasmic Ags might be a general regulatory process, one that is of significant immunologic import.
TAP deficiency is known to alter NK cell development and function in both mice and humans (62
). The altered NK cell function was previously shown to indirectly regulate CD4+
T cell priming in a Toxoplasma gondii
infection model (66
). Hence, it was possible that the several fold increased TH
cell response to the HY alloantigen and listerial Ags in TAP-null recipients were indirectly regulated by NK cells. Therefore, we immunised both wt and NK cell-deficient IL-150
mice with male donor cells and found that the pHY/Ab
response was similar in both recipients (data not shown). Thus, we conclude that NK cells contributed very little to the TH
cell response to HY.
Although TAP and β2-m deficiencies are known to cause ERAD (67
), and ERAD enhances autophagy (55
), we systematically ruled out a role for these degradative processes as mechanisms underlying our central observations. Note that we do not claim that autophagy per se is not involved. But we claim that because TAP-deficiency does not enhance autophagy, the increased class II-restricted Ag presentation in the absence of peptide transport to the ER is not due to overt autophagy. Furthermore, neither β2m nor tapasin deficiencies altered indirect Ag presentation. Their absence, akin to TAP deficiency, renders class I molecules unstable and also results in mice that lack CD8+
T cells. Hence, competition between class I and class II molecules as well as competition between TH
cells and CTL for the same Ag is a most unlikely mechanism by which TAP and ERAAP deficiencies alter indirect presentation of cytosolic Ags.
Our data suggests that TAP and ERAAP are acting directly on class II-restricted cytoplasmic Ags. Such Ags are perhaps processed by the proteasome in the cytoplasm and transported to the ER lumen. Thus, TAP and ERAAP deficiency would prevent transport of processed cytoplasmic peptides into the ER lumen and their subsequent degradation. Such a process would then quantitatively increase the cytoplasmic Ag pool making it available for indirect presentation. Indeed, our data favours this role for TAP and ERAAP in indirect Ag presentation as observed with the increased presentation of the H3ba mHAg by TAP-deficient splenocytes.
Curiously, the effect of TAP and ERAAP on indirect presentation was only observed when both the donor and recipient APC were deficient in the CAP components. Therefore, one possibility is that the donated Ag escapes into the cytoplasm of the recipient APC upon donation by donor allogeneic cells. That such escape might occur is consistent with the need for recipient proteasome for indirect presentation of pHY and the lack of a role for gp91PHOX
for indirect presentation of the same Ag. The escape of Ags from the phagosome to the cytoplasm has been observed with several model and microbial Ags used for mechanistic studies of class I-restricted Ag cross-presentation (68
). Thus, the CAP pathway can sculpt the repertoire of class II-restricted cytoplasmic Ags in both donor and recipient APC.
We view the data obtained with LMP2-deficient mice with caution as prior studies have shown that alterations in the immunoproteasomes can impact CTL repertoire as well as T cell activation (71
). We reported herein that LMP2 deficiency resulted in ~50% reduction in TH
cell response to pHY/Ab
. This result could be explained entirely by deficiencies in T cell repertoire and/or activation in the LMP2-null recipients as suggested in previous reports (71
). Notwithstanding, the TH
response to pHY/Ab
was completely lost when the LMP2-null recipients were immunised with donor cells in which the proteasomes were irreversibly inhibited. If processing of the donated Ag occurred independent of the recipient’s proteasome, then one would have expected the same level of TH
cell response to pHY/Ab
mice were immunised with untreated or epoxomicin-treated donor cells. But instead, the response to the latter was completely lost. Hence, we suggest that the HY alloantigen is donated as an intact protein, which is then processed by the recipient immunoproteasomes for indirect presentation. Furthermore, this finding suggests that the donor HY alloantigen accesses the recipient’s cytoplasm as has been reported for HIV nef and HSV-1 glycoprotein B (68
). This is perhaps why TAP and ERAAP impact indirect presentation of the donated Ags by class II molecules.
It is noteworthy that the inhibition of constitutive and induced HSP90 function in the donor cells disrupted indirect presentation of class II-restricted Ags, and so did the absence of calreticulin. Both HSP90 and calreticulin are implicated as chaperonins for the donation of cross-presented Ags to presenting APC (59
). Therefore, HSP90 and calreticulin may work together to chaperone Ags for indirect presentation of class II-restricted Ags as well. Although calreticulin deficiency could induce/enhance ERAD/autophagy, for afore discussed reasons, these processes do not explain the need for the two chaperonins in indirect presentation. Moreover, calreticulin is also known to act as an “eat me” signal for apoptotic cells, which express the otherwise ER-resident protein at the plasma membrane (73
). Therefore, calreticulin-deficiency may have resulted in poor phagocytosis of the allogeneic donor cells, thereby severely impeding indirect presentation.
Taken together, the model that emerges from the data presented herein is that proteasomes, TAP and ERAAP regulate the quantity of the class II-associated self (mHAgs) and non-self (listerial) peptide repertoire. The increased self-peptide presentation could alter the CD4+
T cell repertoire in recipient cells. Nonetheless, current serological data indicates that the CD4+
T cell repertoire is very similar between wt and TAP-deficient mice (54
). Altered cytosolic Ag pool within donating cells coupled with altered Ag presentation by the APC could explain how the CAP machinery regulates TH
cell responses to indirectly presented cytosolic Ags.