From Immunity and Vaccines to Mammalian Regeneration

Abstract

Our current understanding of major histocompatibility complex (MHC)-mediated antigen presentation in self and nonself immune recognition was derived from immunological studies of autoimmunity and virus-host interactions, respectively. The trimolecular complex of the MHC molecule, antigen, and T-cell receptor accounts for the phenomena of immunodominance and MHC degeneracy in both types of responses and constrains vaccine development. Out of such considerations, we developed a simple peptide vaccine construct that obviates immunodominance, resulting in a broadly protective T-cell response in the absence of antibody. In the course of autoimmunity studies, we identified the MRL mouse strain as a mammalian model of amphibian-like regeneration. A significant level of DNA damage in the cells from this mouse pointed to the role of the cell cycle checkpoint gene CDKN1a, or p21^cip1/waf1. The MRL mouse has highly reduced levels of this molecule, and a genetic knockout of this single gene in otherwise nonregenerating strains led to an MRL-type regenerative response, indicating that the ability to regenerate has not been lost during evolution.

The role of just plain luck and being in the right place at the right time must never be discounted in science. The studies presented in this article arose out of just such personal circumstances. The laboratory of Robert E. Click, at the University of Wisconsin–Madison, pioneered the important role of reducing agents in lymphocyte culture to study T-cell and B-cell collaboration for antibody responses. The laboratory of Darcy B. Wilson, at the University of Pennsylvania, was one of the first to focus on the allogenic histocompatibility responses of T cells in vitro and in vivo. The laboratories of William Paul, Ron Schwartz, and Ethan Shevach, at the Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, were ground zero for T-cell and major histocompatibility complex (MHC) studies. Finally, The Wistar Institute, under the great Hilary Koprowski, who encouraged immunity studies in the context of live viral responses (always with an eye in the direction of vaccines) instead of the more usually studied peptide model antigens, was central to the dissection of immunodominance for vaccines and the search for the effects of autoimmunity on normal biology. It is from this perspective that the studies presented below should be viewed.

RESULTS AND DISCUSSION

Studies in Immunology and Vaccines

After having shown that the antigen-presenting cell, through its MHC class Ia molecule, displayed different antigen-presenting capacities and thus strongly suggested that peptide antigens could bind differently to the MHC molecule [1–5], I came to The Wistar Institute to continue my studies on the recognition of antigen (pigeon cytochrome c as a model antigen) by T cells as defined by the trimolecular complex (Figure 1). The major difference was that the studies at Wistar were directed at multiple disease models instead of a model antigen in which to test the response to self versus nonself and the role of the MHC molecule. The environment that was created was based on Dr Koprowski's focus on (1) neurotropic viruses and related autoimmune central nervous system (CNS) diseases and (2) vaccine production.

For responses to so-called self antigens, we examined the autoimmune response to myelin basic protein, a CNS-derived molecule that could induce encephalomyelitis when injected into rodents and produce disease symptoms with some similarity to those of multiple sclerosis [6–8]. We found T-cell response differences related to the particular MHC recognized that were similar to those we had shown with cytochrome c. Thus, different peptides would stimulate different T-cell clones, depending on the MHC class Ia molecule used. Furthermore, we identified T-cell receptors (TcRs) that were used by pathogenic T cells, which we described as the “V region disease hypothesis” [9–20]. This specifically defined Vβ8, Vα2, and Vα4 as regions used by pathogenic T cells. In addition to these findings and in support of our theory, subsequent genetic mapping of a diabetes susceptibility gene proved to be a TcR V region [21].

For responses to so-called nonself antigens, we examined the T-cell response to herpes simplex virus type 1 (HSV-1) and HSV-2. Dr Koprowski was interested in HSV as a latent virus that might be the cause of multiple sclerosis or other CNS diseases. Nigel Frasier was at The Wister Institute studying HSV latency. Also, studies of the N-terminal 1–23 amino acid antigenic peptide of HSV glycoprotein D (gD) were already ongoing in the laboratories of Bernhard Diestzschold and Elaine DeFreitas at The Wistar Institute, in collaboration with Gary Cohen and Roz Eisenberg at the University of Pennsylvania. We joined the HSV group and began our studies on gD antigen presentation. We found that the N-terminal gD peptides could induce MHC class Ia–related changes in T-cell specificity [22–25]. Thus, responses to self and nonself displayed the same MHC class Ia–specific responses.

Immundominance, the induction of specific peptide responses after priming with a whole protein molecule, was directly related to the specific MHC class Ia molecule used for antigen presentation. We had generated a full set of consecutive gD peptide 20-mers plus a set of peptides with 10 amino acids overlapping between each peptide. We tested multiple strains of mice with different MHCs and found that, after whole gD recombinant protein immunization in complete Freund adjuvant (CFA), each evaluated strain (with a different MHC class Ia molecule) showed different gD peptide responses. Interestingly, none of those peptides were 1–23. We then asked whether infectious HSV-induced immunity gave the same or different responses. To our surprise, all of the peptides could stimulate T cells derived from such mice. Thus, virus infection could overcome immunodominance [26, 27]. Could this be important for vaccine development?

We thought that this knowledge should be useful for the production of a peptide vaccine. There was an interest in the scientific community about using fatty acids to link peptides to cell surfaces. Also, in previous experiments at the National Institutes of Health, we showed that antigens associated with lipid produced strong T-cell responses in the absence of a B-cell response. Could we then produce a vaccine that was specifically T-cell inducing and protective? We first tested peptide alone, and there was no protection against HSV infection. We tested 1–23 peptide coupled to palmitic acid, and it also was not protective. However, 1–23 peptide couple with palmitic acid, when incorporated into a liposome and then CFA, produced long-lived protection against HSV-1 and HSV-2, without a B-cell or antibody response. We found that the best fatty acid proved to be palmitic acid and that all components of the vaccine were necessary. In fact, leaving out any of the components led to suppression of protection. Furthermore, splenic cell transfer of protection was accomplished and required a CD8⁺ population [28, 29].

If the 1–23 peptide as our vaccine construct could induce protection but was not immunodominant, then maybe any HSV peptide in such a construct could induce protection. We had previously produced overlapping peptides along the length of the HSV gD molecule to examine fine specificity and immunodominance. We furthermore made the same peptides with palmitic acid attached. We produced vaccine constructs for the first 6 consecutive peptides attached to palmitic acid, incorporated the constructs into liposomes, and injected them in CFA. These were not immunodominant (ie, injection of gD protein in CFA into BALB/c mice did not induce a response to any of these peptides), but we predicted that all of the peptides would induce protection when configured in our vaccine construct. BALB/c mice were then immunized with each of these 6 peptide vaccines. As seen in Figure 2, some level of protection was seen with every peptide vaccine (US patent 5 837 249).

Creating a functional, strongly protective vaccine that will work in the vast majority of the unselected vaccinated population is both an art and a science. Dr Koprowski, of course, was a master of the craft. However, the best vaccines are still variants of Jenner's cowpox/smallpox killed or attenuated whole virus. With the emergence of human immunodeficiency virus (HIV) and Ebola virus, for instance, the risk of manufacture, let alone administration, of killed or attenuated whole-virus vaccines may be too high for general acceptance. Is it possible to achieve a level of protection equivalent to that of killed/attenuated virus vaccines with a wholly synthetic vaccine that overcomes the limitation of immunodominance? We believe so and have patented a long-since-abandoned generic structure of such a vaccine. Sadly, it was never pursued. However, the science and methods are now free to anyone who wants to use them. Finally and most intriguing is the possibility that antibody might confound the protective response in infections due to pathogens such as HIV and Ebola virus, and this T cell-inducing vaccine might prove significant.

Studies in Regeneration

Out of our studies in autoimmunity, we found that the MRL/lpr mouse, used as a spontaneous model of systemic lupus erythematosus that we were using for drug studies, showed an unusual healing ability. Thus, when we punched ears to number them, instead of providing a long-lived marker, the holes closed up without scarring and virtually disappeared (Figure 3) [30]. This was noticed by Lise Clark, a postdoctoral fellow who came to my laboratory from Will Silvers' laboratory and was yet another person from the Koprowski–Wistar Institute lineage. This ear punch experiment was repeated in MRL/lpr and MRL/MpJ mice, and we realized there was something quite unique about these mice. This was an opportunity we could not pass up.

We explored multiple tissue types in the adult MRL/MpJ mouse for their regenerative ability, including heart [31–33], CNS (such as spinal cord, brain, and optic nerve) [34–36], cartilage [37], and digits [38], and found that this was not just a local effect but was animal wide.

The cells derived from MRL ear tissue also displayed many unique properties. Not surprisingly, cells from the regenerating MRL mouse ears grew quite rapidly, while cells from nonregenerators, including almost all other mouse strains, grew very slowly. An analysis of the cell cycle properties of these cell lines showed that, unlike the slow-growing nonregenerative cells, the regenerative cells displayed a large percentage of the population in the G2 phase, potentially in arrest (Figure 4A and 4B). This suggested that there was an ongoing DNA damage response. In consultation with The Wistar Institute cell cycle guru, Thanos Halazonetis, we examined the protein levels of p53, phosphorylated H2AX, and the DNA repair molecule Rad51, and all were found to be upregulated. But why were we seeing so much stress in these cells?

After Thanos left The Wistar Institute, we approached Paul Leiberman, who was studying Epstein-Barr virus, DNA replication, and telomeres. He had a postdoctoral fellow from Thanos's laboratory, Andy Schneider, who agreed to help along with Paul, and we spent a lot of time talking about what might be going on. First, to further confirm that there was actually DNA damage, we performed a comet assay in which cell lysates were electrophoresed in a gel. Different-sized DNA strands move toward the positively charged end of the gel at different rates, and the level of DNA damage is determined by the size of the tail. As seen in Figure 4C and 4D, the nonregenerative cells showed little evidence of comets (4%), compared with the regenerative MRL cells, which showed 85% of the cells as comets. Such a result was consistent with a G2 arrest interpretation of the cell cycle analysis results. What exactly is happening remains to be determined, but the differences are striking.

The level of damage and potential arrest in G2 suggested that a G1 arrest, which might be expected, was avoided because of a potential lack of a G1 cell cycle checkpoint. Of several G1 checkpoint genes, p15, p16, p21, and p27, the first such gene that was examined was CDKN1a, or p21^cip1/waf1. Cells from nonregenerative B6 mice, the human cell line HCT, and cells from regenerative MRL mice were examined. Western blots showed that p21 was missing from MRL cells, with or without irradiation (Figure 5A). The next step was to determine whether the elimination of p21 would lead to ear hole closure. A p21 knockout mouse (B6;129S2-Cdkn1atm1Tyj/J) made by Tyler Jacks was available from Jackson Laboratories. Cells from the p21 knockout mouse were examined for a DNA damage response (phosphorylated H2AX levels), comets, and the cell cycle pattern, and all showed a response profile similar to that of MRL mouse cells (Figure 5B). Next, we performed ear hole injuries. As we had seen in the MRL mouse, ear hole closure occurred over the same period with the same kinetics (Figure 5C and 5D) [39, 40].

The role of p21 in regenerative healing is not known. However, the lack of p21 could lead to a lack of senescence, reduced levels of transforming growth factor β, and reduced myofibroblast differentiation, leading to reduced scarring. It could also promote a more dedifferentiated state [41, 42]. One interesting model comes out of a study comparing an MRL ear hole closure to a regenerating amphibian limb, performed with David Stocum [42] (Figure 6). The molecule Evi5 (ecotropic viral integration factor 5) is upregulated during the blastema phase (the first 7 days) of regeneration, during which mitosis is low. This is true in the axolotl amputated limb and the MRL injured ear. Evi5, a 110-kDa protein, prevents premature entry into M phase of the cell cycle by binding to and stabilizing Emi1 (early mitotic inhibitor 1). After mitosis and cleavage of the molecule to a 90-kDa form, Evi5 is found in the mid zone and is required for cytokinesis. Another molecule in MRL regenerating tissue is MKLP1 (or Kif23; a candidate regenerative MRL gene), which is also found in the mid zone and involved in cytokinesis. With Emi1 being central to maintaining G2 arrest, the loss of p21 will further promote arrest since p21 has been shown to downregulate Emi1 [43].

This might suggest what p21 is actually doing. Loss of p21 would allow the bypass of the G1 checkpoint. With DNA damage, the cells arrest in G2. This is enhanced by upregulated Evi5 stabilization of Emi1. However, with the loss of p21, Emi1 is further protected from downregulation.

CONCLUSION

The MRL mouse has turned into a deep well of discovery, from its metabolic state, which is aerobic glycolysis [44, 45], to a healing microenvironment similar to that seen in the tumor microenvironment [46–48] but without the development of cancer. Furthermore, genetic mapping studies refined by polymorphic differences have led to the identification of 34 candidate genes on multiple chromosomes [49–54]. One of these genes, Rnf7, is a component of E3 ligase complex, which ubiquinates HIF1a when hydroxylated under normoxic conditions. Its low expression level in MRL mice may be responsible for the high levels of HIF1a found in MRLs [54], which may in turn lead to enhanced aerobic glycolysis [44].

Notes

Disclaimer. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health (NIH).

Financial support. This work was supported by the National Institute of Allergy and Infectious Diseases, the National Institute of General Medical Sciences, the National Institute of Dental and Cranial Research, and the National Cancer Institute (Cancer Center Support Grant CA010815 to The Wistar Institute), NIH; the American Cancer Society; the Harold Y. and Leila G. Mathers Foundation; and the F. M. Kirby Foundation.

Potential conflict of interest. Author certifies no potential conflicts of interest.

The author has submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

References

Author notes