We have recently reported data from this phase I/II randomized safety and dose escalation clinical trial showing that StrataGraft skin substitute is well tolerated and comparable to cadaver allograft in the temporary treatment of debrided wounds prior to autograft placement 21
. This living skin substitute, composed of the NIKS keratinocyte progenitor cell line, was equivalent to cadaver skin in terms of wound bed preparation and did not increase the incidence of wound infection in this patient population. This data, in conjunction with the data presented here, illustrate the clinical utility of StrataGraft skin substitute as a pathogen-free, universal source of human skin in the temporary management of full-thickness skin wounds of several etiologies. As a non-tumorigenic source of long-lived, genetically identical keratinocytes, use of the NIKS keratinocyte cell line is advantageous when compared with other primary keratinocyte samples which senesce after multiple passages. The work described here characterizing the antigenic properties of StrataGraft skin substitute before and after patient exposure supports the continued clinical development of this biologically active skin tissue. The major findings in this study are that StrataGraft skin substitute maintains normal tissue architecture, remains viable after one week in acute, full-thickness wounds, is well tolerated by recipients, and does not induce an acute immune response.
After removal from the patient wound bed the epidermal stratification of the StrataGraft remained intact. A single layer of cuboidal basal cells with suprabasal spinous, granular, and cornified layers were evident in the tissues post-placement, suggesting a lack of acute immune response to and resultant tissue destruction of the allografts. Additionally, keratinocytes within the basal compartment of the stratified tissue stained positively for the cell cycle protein Ki67. Expression of Ki67 has been used as an indicator of keratinocyte proliferation and increases during wound healing.24, 35
Normal proliferative indices (PI; stained cells:total cells) vary based on numerous factors including the anatomical location of the skin and the age of the individual.36, 37
The data shown here indicate that the PI values for StrataGraft skin substitute are more uniform than those for cryopreserved cadaver allograft. These data suggest that StrataGraft is a more consistent source of viable keratinocytes which will respond reliably within the context of a wound bed.
Although they could be found in the wound bed, T cells were only occasionally seen in close apposition to the epidermis, further suggestive of a lack of antigenicity of the allograft. Limited evidence of B lymphocytes in or around the epidermal layers was observed. The significance of Langerhans cells (LC) in the epidermis of both StrataGraft and cadaver allograft tissues after placement in the wound bed is unclear. Historically, LC have been thought to play a role in graft rejection since they express MHC class II and are potent APC.38
However, recent work has suggested that LC may be important in the development of immunological tolerance.39
While the exact role that LC play during transplantation may still be debated, it is clear that these cells traffic rapidly into and out of the skin and their presence in StrataGraft skin substitute indicates that StrataGraft tissue readily allows cellular trafficking. In sum, there was no evidence of rapid infiltration of leukocytes including T and B lymphocytes nor was there evidence of rapid destruction of the allograft architecture which is seen when there is significant neutrophilic or mononuclear cell influx and activation seen during acute inflammatory responses. Over the years, many studies have been focused on determining the immunogenic elements of allogeneic tissue responsible for acute graft rejection. Studies have shown that passenger leukocytes are largely responsible for this response40, 41
and that the rejection of transplanted tissues can take place either directly or indirectly. Acute rejection most often occurs as a result of direct allorecognition in which intact MHC class I and II molecules on the allografted tissue are recognized by the recipient T cells. This type of direct response tends to be quite rapid and the transplanted tissue is quickly recognized, infiltrated by recipient leukocytes, and broken down. Indirect allorecognition occurs when transplanted donor cells die and their MHC class I or II molecules are processed and presented on the recipient antigen presenting cells (APC). This type of alloresponse tends to occur more slowly and has been shown to play a primary role in chronic tissue rejection as well as a lesser role in acute immune reactions.42, 43
Previous work has shown that the incubation of allogeneic human tissue in vitro
prior to transplantation reduces its immunogenicity. This is likely due to the death of passenger leukocytes during the culturing period, which reduces or eliminates acute allorecognition responses.44-46
StrataGraft skin substitute does not contain leukocytes or dendritic cells and is therefore does not generate a direct alloresponse in the recipient.
Human skin is constantly exposed to a tremendous number of environmental antigens and the presence of immunological markers on the keratinocyte surface is directly affected by the microenvironment in which these cells exist. Once a cutaneous wound has occurred, the milieu of pro-inflammatory cytokines may provoke keratinocytes to become active participants in the cutaneous immune response. The expression of MHC class I and class II antigens, in particular, HLA-A, -B, and -DR, have been closely associated with tissue rejection in humans.15, 16
The ability of keratinocytes to present antigen to CD4+
T cells is tightly controlled through the regulated expression of HLA-DR and essential co-stimulatory molecules on their surface. Previous work has shown that the potent pro-inflammatory cytokine IFN-γ is able to induce HLA-DR expression in keratinocytes within a cultured skin substitute.47
The data presented here indicate that both NIKS and primary NHEK tissues lack constitutive expression of the MHC class II antigen HLA-DR. In vitro
exposure of the tissues to IFN-γ was sufficient to enhance the expression of HLA-ABC and induce the expression of HLA-DR and the co-stimulatory molecule, CD40, similar to that seen in other skin substitutes.47
However, even at high concentrations of IFN-γ, expression of the key co-stimulatory molecules B7-1 or B7-2 was not detected.
In contrast to what one may anticipate during an acute inflammatory response and unlike that seen in animal models of skin grafting48, 49
, placement of StrataGraft skin substitute in acute full-thickness wounds in human patients is not sufficient to induce the expression of HLA-DR on the allografted keratinocytes. The HLA-DR expression seen in the tissues was instead suggestive of the presence of dendritic cells and leukocytes. This lack of HLA-DR expression by the keratinocytes coupled with the lack of induction of key co-stimulatory molecules supports similar findings in the literature and suggests that NIKS cells are incapable of delivering the co-stimulatory “second signal” essential for activating T cells even in the presence of IFN-γ.50-52
Furthermore, this result is in agreement with clinical studies of other skin substitutes which indicate a lack of clinical rejection.53-55
Several cutaneous skin disorders are associated with aberrant expression of HLA-DR on the keratinocyte surface, leading to increased infiltration of inflammatory cells.56, 57
The results presented here indicate that NIKS cells do not express HLA-DR after placement in acute wounds and are therefore unlikely to act as antigen presenting cells leading to acute rejection and enhanced inflammatory responses.
While this study did not investigate the role of minor histocompatibility antigens, the inability of NIKS keratinocytes to induce expression of co-stimulatory molecules most likely limits their ability to engage in functional T cell stimulation and induce direct allorecognition. However, indirect allorecognition is an ongoing process due to the continual trafficking of recipient APC through the graft and the turnover of allograft cells. Our data indicate that after placement in full-thickness skin wounds, HLA-ABC was expressed in the majority of stratified keratinocyte layers. We therefore anticipate that like other allogeneic skin substitutes, StrataGraft tissue will not persist permanently on an immunocompetent recipient but will be slowly replaced as the patient’s own keratinocytes are able to proliferate and fill in. This is in agreement with previous studies of the Apligraf skin substitute, which showed that it does not permanently engraft and is undetectable after 2-3 months.58-61
Results from in vitro
cellular assays indicated that the patients enrolled in this trial did not develop enhanced proliferative responses to NIKS cells or to allogeneic PBMC after exposure to StrataGraft skin substitute. These data support the conclusion that exposure to StrataGraft tissue does not prime the patient PBMC in vivo
. Likewise, no evidence of NIKS-directed cytotoxicity after exposure to StrataGraft tissue was found. It is well established that susceptibility to NK cell lysis is inversely proportional to MHC class I molecule expression on target cells62-65
, thus the presence of HLA-ABC molecules on NIKS keratinocytes likely provides protection from cellular lysis by NK cells.66, 67
It is likely that the constitutive presence of MHC class I molecules and the lack of MHC class II and co-stimulatory molecules reduces the recognition of the allogeneic NIKS keratinocytes as foreign by the patient immune system. In addition, no correlation was observed between increased StrataGraft skin substitute dose and elevated PRA values, nor did patients develop antibody responses to cells of the StrataGraft skin substitute. These in vitro
functional assays are indicative of a lack of recipient sensitization to or acute rejection of StrataGraft skin substitute tissue.
Composite human skin substitutes such as those created using the organotypic culturing method outlined here have been suggested to be immunologically neutral, thus providing promise for their acceptability in a grafting environment.50, 55, 68, 69
While LC are considered the primary APC within cutaneous tissues, it has been established that endothelial cells are also capable of presenting antigen.70
The tissues described here are devoid of these cell types. Furthermore, we have shown that NIKS keratinocytes do not upregulate co-stimulatory molecules B7-1 and B7-2, thus limiting the possibility of successful T cell activation even in the presence of inflammatory signals such as those found in the wound environment. Beyond IFN-γ, other immunoregulatory cytokines including IL-1α, IL-6 and IL-12 are insufficient to elicit an alloresponse against NHEK or dermal fibroblasts in functional in vitro
assays, further suggestive that keratinocytes can generate an inert skin substitute.47
StrataGraft skin substitute has unique qualities that make it an invaluable resource for use in epidermal development, tissue engineering and transplantation studies. The work presented here shows that StrataGraft tissue generated from NIKS keratinocytes is well tolerated, and does not elicit an acute immune response in human graft recipients. These findings further substantiate the utility of this unique cell-based product for therapeutic applications in the treatment of cutaneous wounds and disease.