Summary of allograft rejection and immunological memory in adult
1. Introduction
Over the last few decades, the amphibian
The objectives of this review are first to provide short background information on the immune system and skin graft biology in
2. The immune system of Xenopus laevis
The frog
The additional developmental transition occurring during metamorphosis in
Thus, the larval and adult
A major attribute of the
3. Skin graft rejection in adult Xenopus laevis
Skin graft rejection is a well-established technique in
We have recently described this technique in details (Nedelkovska, Cruz-Luna et al. 2010). Succinctly,
3.1. Conserved role of T cells in skin graft rejection
In adult
The strict T cell dependency of skin rejection has been clearly established in
In order to assess more directly the role that CD8 T cells play in these responses
To further characterize the CD8 T cells involved in graft rejection, our lab adapted a whole-mount immunohistology technique. This procedure allows us to visualize lymphocytic infiltration into unfixed transplanted skin tissues using fluorescent antibodies (Ramanayake, Simon et al. 2007). Additionally, this method preserves the tissue structure and we can use several antibodies conjugated to different fluoropores to see exactly where cells are located and distinguish what kind of cells are present in the grafts. Therefore, this technique is a powerful tool which we can use to characterize and monitor immune effector cells mediating the immune responses of
Using whole-mount immunohistology we found that, unlike isograft controls, MHC-disparate grafts that were undergoing rejection were infiltrated with a large number of CD8 T cells. These CD8 T cells were mainly distributed in areas where the graft was not yet rejected and the silvery irridophores were still persisting. Moreover, there was an inverse correlation between the percent rejection and number of infiltrating cells. For example the most prominent CD8 T cell infiltration occurred at day 7 when there was only 50% rejection. Additionally, these grafts also had significant infiltration of class II positive cells which were more numerous than the CD8 T cells. As mentioned before all adult leukocytes have class II expression in adult
As previously discussed, the main difference between MHC-disparate and minor H-Ag-disparate grafts is the time it takes for complete graft rejection, which is either acute or chronic, respectively. Therefore, one might assume that this is due to the lesser number of infiltrating effector T cells since only minor H-Ags are involved in these responses. On the contrary, however, we found that minor H-Ag-disparate grafts were infiltrated by similar numbers of both CD8 and class II positive cells, but with delayed kinetics (Ramanayake, Simon et al. 2007). In these minor H-Ag-disparate allografts the peak of immune cell infiltration was also observed when the graft was about 50% rejected which in this case occurred 15 days after transplantation, rather than 7 days as in the case of MHC mismatched grafts (Table 1).
Whole-mount immunohistology is a very powerful technique to study infiltration of immune cells; therefore, we are currently using different antibodies such as
3.2. Characterization of the immunological properties of heat shock proteins (HSPs) using skin graft rejection
Our
As mentioned, the LG-6 and LG-15 clones share the same MHC haplotypes but differ by minor H-Ags, and frogs primed with a first set of minor H-Ag-disparate skin graft reject a second set skin significantly faster. Furthermore, this accelerated rejection is thymus-dependent and Ag specific (e.g., a third party skin graft rejection is not accelerated). This system has revealed to be ideal in investigating whether the ability of hsps to generate CD8 T cell responses is conserved between mammals and amphibians.
We found that if we first immunize LG-6 clones with either gp96 or hsp70 purified from LG-15 liver (meaning that those hsps would carry LG-15 minor H-Ags) and then graft them with an LG-15 allograft, the graft undergoes accelerated rejection in comparison to control unimmunized animals or animals that were immunized with LG-6 derived hsps (carrying self-Ags) (Robert, Gantress et al. 2002). Additionally, syngenic grafts were never rejected regardless of the immunization status of the animal, which rules out possible Ag-independent pro-inflammatory effect induced by the hsps. Furthermore, these
Type of skin grafts¶ |
|
|
|
Rejection at 21°C | 30 - 100 | 18 - 22 | 18 - 22 |
Peak CD8 T cell infiltration | 15 | 7 | 7 |
Rejection after priming with same first set graft | 16 – 30 | ND | ND |
Rejection after gp96 or hsp70 immunization |
20 - 30 | ND | ND |
In mammals it is known that immune responses generated by hsps are mediated by CD8 cytotoxic T lymphocytes (CTLs). Using an
To explore
To test the effector function of adoptively transferred CFSE+ cells we monitored skin graft rejection. We found that unimmunized frogs carrying a minor H-Ag-disparate graft had accelerated skin graft rejection which reached 45-90% by day 10 after adoptive transfer of splenocytes (Maniero and Robert 2004). On the other hand, animals that carried isogenic grafts did not show signs of rejection. These rejection kinetics are reminiscent of a secondary T cell mediated rejection that occurs with animals primed with gp96 carrying minor H-Ag complexes. This means that gp96 is able to prime CD8 effector T cells that when adoptively transferred can recognize minor H-Ags presented
3.3. Possible role of nonclassical MHC class Ib genes in skin transplantation
As discussed above, CD8 T cells are critically involved in skin graft rejection in
4. Immune responses and tolerance to skin antigens during larval and metamorphic stages
Of particular relevance for the immunological aspects of tissue transplantation, is the fact that unlike mammals, the
In this section we will discuss the ability of
Another possible source of conflicting results concerns development, especially metamorphosis, which may play the most important role in generating tolerance. Metamorphosis in frogs, both initiation and completion, is under the control of the thyroid hormone (Furlow and Neff 2006; Tata 2006). The thyroid hormone starts being produced around stage 50, peaks at stage 60, and the levels are back to normal at the end of metamorphosis (Figure 3). Also there are two different thyroid receptors- α and β. Thyroid receptor-α is expressed early on after hatching while β comes up concomitantly with the thyroid hormone (Figure 3). In addition, the action of the thyroid hormone during metamorphosis is local and not systemic. This means that in certain tissues the changes associated with metamorphosis may start earlier than in other tissues and this may directly impact the ontogeny of tolerance. The complexity of the metamorphic transition is likely to result in marked individual variation even in clonal animals, including the differences in
the immune system. This is indeed observed by the individual variation in morphological changes (e.g. there are easily several day differences in the time of complete tails loss in cloned progeny).
In order to address some discrepancies in the literature we will first discuss the induction of tolerance to minor H-Ag followed by MHC-disparate grafts. The mechanism(s) as well as possible effector cells involved in allotolerance will also be considered. Finally, we will provide some perspective on using new tools and methodologies to more conclusively answer questions associated with tolerance.
4.1. Immune response and allotolerance in X. laevis larvae to minor H skin antigens
In contrast, during metamorphosis (which actually includes the time spanning 15 days before (st. 58) and about a month after metamorphosis, perimetamorphic animals) up to 50% of the sibling grafts were actually tolerized (i.e., not rejected), and those that were rejected followed a very slow rejection kinetics (20 - 80 days for complete rejection) in comparison to grafts on young larvae or adults (10 - 20 days). This suggests that metamorphosis is a special developmental time during which immune tolerance can be induced. This could be due to the fact that the immune system is undergoing complete remodeling and there is reduced number of lymphocytes. For example, more than 50% thymocytes die during metamorphosis (Du Pasquier and Weiss 1973). However, several pieces of evidence do not support the hypothesis that tolerance induction at metamorphosis is due to an insufficient number of lymphocytes. For instance, regardless whether the grafts are rejected or tolerized they are infiltrated by lymphocytes (Horton 1969; Bernardini, Chardonnens et al. 1970), while autografts are not. This implies that the grafts are recognized as non-self although they are not rejected. Also if tolerance is induced due to the lack of lymphocytes one would assume that once metamorphosis is completed and the number of lymphocytes is recovered, these grafts would be rejected. However, that is not the case since these grafts survive for more than two years. Interestingly, this tolerance can be broken by a third party graft (Bernardini, Chardonnens et al. 1970), which suggests that some H-Ags may be shared between the two different grafts. Therefore, once a T cell response is initiated against the third party graft that response can also be cross-reactive to the tolerized graft. Thus, it appears that during metamorphosis
On the other hand, allograft tolerance capacity was found to occur also at premetamorphic stages (Table 2) when more genetically defined and homogeneous animals were used (DiMarzo 1980; DiMarzo and Cohen 1982a; DiMarzo and Cohen 1982b). Remarkably, both pre and perimetamorphic larvae of inbred strains were able to induce tolerance against allografts that were either minor H locus or even MHC-disparate, whereas all postmetamorphic froglets rejected 100% of the grafts (Table 2). In addition, differences in rejection depending on the developmental stage were noted; in general younger larvae (st. 47/48) had a higher propensity for becoming tolerant while older larvae (st. 57/58) had the ability to reject more grafts. In either case, the grafts were rejected with delayed kinetics in comparison to postmetamorphic animals. Despite the discrepancy on the ontogeny of allotolerance in
4.2. Immune response and allotolerance in X. laevis larvae to MHC-disparate grafts
Although early experiments have indicated that during metamorphosis allotolerance is induced to minor H-Ags (Chardonnens and Du Pasquier 1973; Chardonnens 1975; DiMarzo 1980; DiMarzo and Cohen 1982a; DiMarzo and Cohen 1982b), other work has revealed that the immunogenetics of tolerance are complex. Family studies using field-collected outbred adult
In order to bypass undefined genetic variation, Barlow et al. (1981) used MHC defined inbred strains of
In addition to the genetic background of the grafts, as mentioned before, graft size plays an important role which can tip the balance between rejection and tolerance (Bernardini, Chardonnens et al. 1970; Chardonnens and Du Pasquier 1973; Barlow and Cohen 1983). In general smaller grafts (1-2 mm2) are more readily rejected (can reach up to 90% rejection) in perimetamorphic animals when there is a one MHC haplotype difference between donor and host. On the other hand, larger grafts (4-9 mm2) almost always induce tolerance. The same trend holds true for grafts differing by two MHC haplotypes except the capacity for rejection is greater since tolerance is not easily induced in this case.
4.3. Mechanisms of allotolerance
Cell transfer approaches were developed to investigate the cellular mechanism responsible for tolerance induction and maintenance of skin Ags in
In a complementary set of experiments, adoptive cell transfer was used to determine if tolerance to skin minor H-Ags can be broken (Du Pasquier and Bernard 1980). Splenocytes from isogenic adults primed by minor H-Ag-disparate skin grafts were adoptively transferred into isogenic metamorphic recipients with tolerized skin graft genetically identical to the one used to prime the adults. This adoptive transfer of primed anti-minor H-Ag lymphocytes was not able to break tolerance in animals that tolerated the grafts for at least six months. However, if a second graft, identical to the tolerized graft, was placed at the time of the adoptive transfer, an acute rejection of this graft was initiated but stopped within 10 days. The result was a graft that was half rejected and half healthy. This suggests that even though the animal is tolerant to the allograft, the primed transferred cells are able to cause graft destruction although the number and/or survival of these reactive cells is not sufficient to complete the rejection. Presumably, if another transfer was done the graft may be fully rejected. Another possibility is that a subset of regulatory cells home to the skin at the time of transplantation to induce and maintain tolerance or suppression, and that the presence of adoptively transferred anti-minor H-Ag lymphocytes prevent or delay the migration of these regulatory cell in the transplanted skin. Taken together, these data strongly suggest that tolerance in larvae does not depend upon deletion of alloreactive cells but rather is maintained by suppressor or regulatory cells that can be adoptively transferred.
The preponderant role of T cells in larvally-induced allotolerance has been established by thymectomy. When larvae were thymectomized during early larval life before the migration of T cell precursors into the thymic anlage, graft rejection of premetamorphic larvae as well as postmetamorphic froglets was severely impaired (Table 2) demonstrating that without the thymus alloreactive cells can not develop (Horton and Manning 1972; Barlow and Cohen 1983; Kaye and Tompkins 1983; Nagata and Cohen 1983). Furthermore, the impaired rejection capacity of premetamorphic larvae that were thymectomized at early developmental stage can be rescued by implantation of an intact larval or adult thymus (Arnall and Horton 1986). When these T cell deficient thymectomized larvae are implanted with an isogenic thymus their capacity to reject both minor H-Ags and MHC-disparate skin grafts was fully restored. However, when thymectomized larvae were reconstituted with a thymus that was either MHC-disparate or differed even by minor H-Ags from the host, rejection was restored only to grafts that were MHC incompatible both to the host and the thymus donor. Interestingly, these reconstituted animals had an impaired ability to reject minor H-Ag-disparate grafts. Notably, when MHC incompatible thymi were transplanted, skin grafts from the same donor were tolerated even though they were able to elicit proliferative responses against the thymus donor cells in an
In contrast to early thymectomy, late larval thymectomy did not have an effect on rejection (Table 2). Interestingly, however, thymectomy performed during mid-larval stages significantly impaired the ability of animals to become tolerant to allografts (Table 2) (Barlow and Cohen 1983). Moreover, this impairment was dependent on the number of different MHC haplotypes as well the size of the grafts.
In conclusion, it is clear that
|
|
|
|
Developmental Stage | 47 - 58 | 58 – 1 month post metamorphosis | 2 months post metamorphosis |
Minor-H-Ag disparate grafts | Tolerance induction |
Tolerance induction |
Rejection 100% |
1 MHC haplotype disparatre grafts | Tolerance induction |
Tolerance induction |
Rejection 100% |
2 MHC haplotypes disparate grafts |
Rejection | Rejection MST 42 |
Rejection MST 18 |
Early larval life thymectomy | Impaired graft rejection | No effect | Impaired graft rejection |
Mid larval life thymectomy | No effect | Impaired tolerance | No effect |
Late larval life thymectomy |
No effect | No effect | No effect |
*Discrepancy between different studies. Chardonnens and Du Pasquier (1973) reported that premetamorphic larvae were capable of rejecting grafts similary to postmetamophic froglets while several reports from the Cohen lab reported that premetamoprhic larvae (as early as stage 47) had the capacity to induce tolerance.
+, low incidence of tolerance induction
+++, high incidence of tolerance induction
MST: Mean Survival Time (in days)
4.4. New tools to study tolerance
During metamorphosis, as described before,
There is still much unknown regarding the mechanism(s) associated with suppression during allotolerance. For instance, we still don’t know which effector cells are involved, even though we speculate that they might be T regulatory cells. The mode and site of action of these regulatory cells is also not defined. Using some newly generated tools and techniques we can start to answer some of these questions. Initially, it will be of great interest to immunize perimetamorphic larvae with hsps carrying either MHC or minor H-Ags and then look for immunological memory toward skin grafts (same genetic background as the Ags carried by the hsp) transplanted post metamorphosis. These experiments will show if the Ags alone are capable of generating tolerance by possibly negatively selecting reactive thymocytes in the thymus or by generating specific T regulatory cells toward those particular Ags.
To further investigate the effector cells involved in these responses, we will be able to use transgenic animals. Transgenesis in
As mentioned in the introduction the genome of the
Another area of investigation where
5. Conclusion
The studies reviewed here highlight the versatility and attractiveness of
Acknowledgments
We would like to thank Nikesha Haynes and Juan Oves for critically reading the manuscript. Also we would like to acknowledge the X. laevis research resource for immunobiology (
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Notes
- All animals were handled under strict laboratory and UCAR regulations (Approval number 100577 / 2003-151), minimizing discomfort at all times.