This study was prompted by an expanding literature concluding that memory T cells, but not naïve T cells, preferentially survived in an MHC-deficient environment [2
]. We initially predicted that such an environment would provide a means to enrich autoreactive cells and study their intrinsic properties, for example, by ‘parking’ diabetogenic NOD T cells in an MHC-less NOD environment (the NOD DKO model). We also sought to use this model to explore potential differences between CD4 and CD8 T cell survival under these conditions, to address the role of MHC in desensitizing (‘tuning’) or in sensitizing T cell responses against self-antigens, and to begin to address how intrinsic negative regulatory factors influence this process.
The results of adoptive transfer of NOD T cells into the MHC-deficient NOD environment supported our expectations, as the majority of donor T cells died within four weeks of transfer into NOD DKO animals, but the few T cells that remained were associated with fulminant destruction of syngeneic, MHC-positive NOD islet grafts. Islet destruction was not simply due to T cells undergoing HP in the lymphopenic recipient, since it did not occur in the sixty-day window when we adoptively transferred T cells into lymphopenic SCID mice. Nor could this be explained simply by use of T cells from an autoimmune prone donor, as the results of experiments in the non-autoimmune prone B6 background were essentially indistinguishable from the results in the NOD background. Finally, graft rejection was probably not due to alloreactivity, as it was not apparent in single MHC knockout recipients. While we cannot completely rule out the possibility that this is because of a requirement for cooperative interactions between CD4 and CD8 T cells subsets, the response appeared to hinge on the concurrent absence of Class I and Class II MHC.
We used a syngeneic tumor model [49
] as a complementary model to examine how loss of self influenced T cell survival and activation. The B6 DKO model recapitulated results from single class I and single class II knockout mice with respect to T cell survival [17
]. In addition, naïve T cells delayed LL tumor growth in the MHC-deficient environment, and this effect was enhanced when we used antigen-experienced cells from tumor bearing donors (i.e
., they were somehow ‘primed’), even though the donors themselves were unable to reject the tumors. The observation that tumor rejection did not occur in MHC-positive, lymphopenic SCID animals that were adoptively transferred with naïve T cells, or with antigen-experienced T cells that did not reject tumor in the donor (AE-NR), indicates that absence of MHC was essential to uncover the tumor reactive population, i.e
., HP in the MHC-replete environment alone could not provide sufficient impetus for activation of quiescent self-(tumor) reactive T cells. Furthermore, failure to reject tumors was not due to infiltration by suppressive double negative (CD4−/CD8−) tumor infiltrating lymphocytes as has been reported for other syngeneic murine tumors [52
On the other hand, T cells that were deliberately primed under conditions of productive inflammation (by ectopic expression of FasL in tumors), were able to destroy secondary tumors even when MHC was present, indicating that the functional phenotype of memory T cells generated by canonical innate immune responses or by exposure to exosomes harboring apoptotic bodies (such as the seen with AE-R T cells) behaves differently than that generated by HP (i.e., by transfer of naïve T cells or AE-NR T cells to SCID mice).
It is worth noting that there were three distinct points of event-free survival for the groups tested. First, it took 10–11 days for 50% of wild type B6 mice, B6 DKO mice (without T cells) or any of the SCID groups (with or without T cells) to develop tumors, and 100% of mice had tumors 14 days after challenge, although the rate of tumor growth in SCID mice that received unchallenged T cells or AE-NR T cells was slower than it was in mice with no T cells. In contrast, it took 14 and 21 days, respectively, for 50% of B6 DKO mice that received unchallenged T cells and B6 DKO mice that received “AE-NR” T cells to develop palpable tumors (significantly different from controls to p<0.04 and p<0.01). Moreover, while tumor growth was significantly slower in DKO mice that received unchallenged T cells than in the control or SCID groups, the slowest tumor growth rate was recurrently observed in the “AE-NR” B6 DKO recipients.
The sum of our results suggests that complete absence of MHC (class I and class II) incites T cell autoreactivity, or rather, removes a component of intrinsic negative regulation that maintains tolerance to self. There are conflicting data regarding loss of MHC and T cell reactivity (reviewed in [33
]). Several reports indicate that self-interactions (TCR-MHC) maintain a high threshold for self-antigens [27
], whereas others show that these interactions promote modest receptor clustering and CD3ζ chain phosphorylation [20
] and in the case of CD4 cells, MHC class II availability promotes activation of Rap1 and Rac1, enhancing motility [65
], suggesting self-interactions are responsible for partial activation that sensitizes the TCR as well as for increasing the potential for these cells to encounter and respond to antigen. One possibility that remains to be explored is that quantum signaling through the TCR might be responsible for sensitization and for tuning in T cells with varying avidity for self-peptides. Quantum signaling refers to the difference in signal magnitude that can be achieved by the number of receptors that are bound by ligand (determined by receptor density and ligand concentration) and the temporal occupancy of each receptor (determined by affinity)
The phenotype was dependent on loss of both MHC class I and MHC class II, as it was not evident in the single knockout mice. Unlike our results in the single class II knockout mice that did not reject pancreatic islets after adoptive transfer with naïve T cells, Bhandoola and colleagues reported rejection of MHC-positive skin grafts in approximately one third of MHC Class II-deficient animals that received naïve CD4 T cells [27
]. Given that skin is particularly sensitive to T cell-mediated attack, rejection of only a fraction of the grafts suggests that the response unleashed by the absence of MHC class II alone was not very robust (as one might expect if the response is derived from T cells bearing low affinity receptors for self), even though lymphocytic infiltration into the skin was observed in all the animals.
Our results showing survival and autoreactivity of adoptively transferred, naïve CD4 T cells resembled those reported for the skin graft experiments into MHC class II SKO mice [27
], with two of five B6 DKO mice that were adoptively transferred with naïve T cells showing delayed tumor growth for >50 days, and all of the mice examined containing CD4 T cell infiltrates in the tumors. In contrast, both CD4 and CD8 T cells survived in the MHC-deficient environment after they were exposed to tumor in the donor (antigen-experienced), and both the CD4 and the CD8 populations infiltrated LL tumors in the recipients. Hence, the results support current dogma that CD4 T cells persist longer than CD8 T cells in an MHC-deficient environment [20
], and the improved outcomes seen in mice that received antigen-experienced T cells were expected because these would contain MP CD8 T cells that are less dependent on MHC for survival.
Unlike what we observed in MHC-positive SCID mice, thus, unchallenged T cells and antigen-experienced T cells from donors that did not reject the tumor challenge could be induced to destroy LL cells following adoptive transfer into the MHC-deficient environment. This indicates that absence of MHC released a previously quiescent population with effector potential that was controlled by the presence of MHC in the unchallenged donors, and it also released a tumor sensitized (antigen-experienced) population that had been controlled by the presence of MHC in donors that had been challenged with, but failed to reject tumors. This latter conclusion is possible both because of the delay in the median survival and the rate of tumor growth.
These observations could be explained by release of T cells from intrinsic negative regulation in the absence of MHC. In other words, the same mechanisms that restrain T cell proliferation might control “tuning”. Among various candidates, we elected to study the effects of Tob1, which acts to silence the IL-2 promoter [66
], and also modulates the activity of SMAD transcription factors, which are responsible for most of the anti-proliferative effects of transforming growth factor-β (TGF-β) [66
]. CDK inhibitors including Ink4 proteins and p27 are upregulated by TGF-β [67
], and at least p27 also is strongly induced by Tob1 [14
]. The data from adoptive transfers using Tob1-deficient cells were initially counterintuitive, as the recipients showed no propensity to reject syngeneic tumors and the resulting peripheral T cell phenotypes were reversed from mice that received wild type T cells. Tob1-k/o CD8 cells were able to persist in spleen and draining lymph nodes in MHC-deficient recipients, suggesting that Tob1 deletion was sufficient to overcome the requirement for MHC of naïve CD8 T cells (and thus these cells survived in both DKO and class I SKO mice), whereas CD4 cells did not tolerate the simultaneous loss of MHC and Tob1.
The response of naïve and memory cells to a lymphopenic environment has been the subject of several recent studies. Under normal conditions of HP, memory cells have an advantage due to their lower threshold of response for IL-7, clonal competition, and reduced sensitivity to the effects of Treg cells. It is reasonable to assume this balance would be even more noticeable in the MHC-deficient environment where there are additional obstacles that impair survival of naïve T cells. However, the ability of naïve T cells to undergo HP is different when there are abundant levels IL-2 and IL-15. Not only do these cytokines allow for expansion of naïve cells, but they also generate functionally competent cells [2
]. The peripheral polyclonal T cell populations from donor mice in our experiments did not contain appreciably changed frequencies of CD44hi
cells that might reflect an alteration in phenotype dictated by the immune status of the donor. Nor were the ratios of CD4/CD8 subsets altered, an expected result given that the T cell enrichment approach was a negative rather than positive selection approach, but the origin of the expanded population cannot be ascertained from this fact as the cells acquired a memory-like phenotype in the recipient mice.
Finally, we cannot completely exclude a role for CD4/CD25/FoxP3+ Treg cells. Independent studies indicate Treg cells are deficient in MHC-less recipients [27
], and our preliminary data suggest that the MHC-deficient environment does not strongly support survival of Treg cells. We quantified FoxP3 cells in samples from the mice a posteriori
, and thus relied on immunohistochemistry of formalin-fixed and paraffin embedded tissues. No difference was apparent in SCID mice that received naïve, AE-NR, or AE-R T cells or in DKO mice that received naïve or AE-NR T cells. The absence (or loss) of Treg cells may reflect their formation in the thymus at the boundary of TCR affinities for self, separating cells that are high affinity and subject to negative selection from those that are low affinity and subject to positive selection. The proclivity of Treg cells for self probably makes them particularly dependent on MHC, both for survival and for tuning; thus, they might die readily in a peripheral environment that is devoid of MHC.
Nevertheless, if regulatory T cells were the main reason why tolerance was maintained in adoptively transferred SCID mice, as compared to DKO mice, we might expect them to similarly dampen anti-self (tumor) responses in animals that received antigen-experienced T cells primed in the presence of FasL (AE-R), since survival of Treg cells should be assured in the MHC-positive SCID environment. Similarly, while the observation that class I SKO did not universally reject pancreatic islet grafts could be consistent with survival of Treg cells in the MHC class II-positive environment, the prediction would then be that Treg cells should die in class II SKO mice and that these animals would reject islet grafts, and this was not the case. Hence, the sum of our data implicates intrinsic mechanisms that are at least partially independent of Treg cells for the generation of islet and tumor rejection phenotypes in DKO mice.
In summary, our results are consistent with the interpretations that MHC class I is required for survival of naïve CD8 T cells, but not memory CD8 T cells, and that MHC class II provides ‘tuning’ signals that restrain autoreactivity by CD4 T cells. It is widely assumed that most tumors express only self-antigens and are therefore immunologically silent. Hence, unresponsiveness to tumors, or to pancreatic islets in non-autoimmune prone animals, is reversed by the absence of ligands that limits self-reactivity, i.e., self-MHC.