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Literature Review | Skin Grafts and Dendritic Cell–“Enhanced” Humanized Mouse Models in the Preclinical Evaluation of Therapeutic Cancer Vaccines

Vaccines represent one of the most successful medical advances in human history, with prominent examples including widely administered vaccines against hepatitis B virus, human papillomavirus (HPV), and severe acute respiratory syndrome coronavirus 2. Improved preclinical mouse models that can recapitulate disease features and predict human responses are essential for overcoming the limited immunogenicity and efficacy of current cancer vaccines, thereby enhancing their clinical applicability and therapeutic effectiveness.

Among these models, humanized mice—engineered to engraft human immune cells and harbor human or human‑like tumors—represent a valuable experimental tool. Among the immune cells involved in the immune response, dendritic cells are currently recognized as the most potent antigen‑presenting cells and, in the near future, are poised to become another powerful weapon in the fight against cancer.

In this study, the authors investigated the impact of HLA matching in the NSG system, as well as complementary strategies involving human peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs), to develop two novel humanized mouse models. These models were used to evaluate the immunogenicity and therapeutic efficacy of vaccines targeting human papillomavirus (HPV16) antigens and delivering tumor antigens to human CD141+ dendritic cells (DCs). The models are suitable for characterizing the immunogenicity and therapeutic effects of vaccines that target HPV antigens expressed in dysplastic skin or that deliver diverse tumor antigens to human Clec9A+ DCs in vivo.

Research findings

Result 1: Immune status of NSG-A2 mice following PBMC transplantation

Two weeks after transplantation, the proportion of human-derived immune cells in fresh PBMCs increased from 20% to 80%, compared with that in cryopreserved PBMCs.

Twenty-eight days after transplantation, the engraftment rate remained above 70%, with a pronounced post‑engraftment expansion in the spleen, increasing from 20% in the first week to 80%. Peripheral blood PBMCs gradually engrafted in peripheral organs and/or underwent passive expansion within specific cell populations in those organs.

Bone marrow cells (CD11b+), DN cells, NK cells, and NKT cells show decreased abundance in the spleen and blood; B cells decrease in the blood but increase in the spleen.

T cells constitute the largest population; CD4+ T cells remain comparable to those in the donor, whereas CD8+ T cells are significantly elevated relative to the donor. The abundance of double-positive T cells increases over time, a phenomenon associated with the onset of graft-versus-host disease (GvHD).

Spleen cells from mice 21 days post-transplantation were stimulated with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, and intracellular IFNγ accumulation was assessed by flow cytometry to evaluate the immune response.

Both CD4+ and CD8+ T-cell subsets express high levels of effector cytokines, demonstrating their capacity to respond to T-cell receptor–mediated stimulation within 21 days after transplantation.

Conclusion:

Fresh PBMC transplantation yields superior outcomes; from day 7 to day 28 post‑transplant, both peripheral blood and splenic hCD45+ cells show significant increases, with the splenic response slightly delayed. In vitro validation of immune responses demonstrates that, within 21 days after transplantation, the graft retains the capacity to respond to T‑cell receptor–mediated stimulation. IFN‑γ levels are markedly elevated in both CD4+ and CD8+ T cells.

Result 2: A humanized mouse skin transplantation model and its response to a DNA-HPV therapeutic vaccine.

The authors first established a humanized mouse skin transplantation model.

Following hybridization, the F1 generation exhibited characteristic thickening of the epidermis in NSG-A2_K14E7 skin.

The F1 NSG-A2_B6/K14E7 donor was subjected to lethal irradiation, and skin from the F1’s ear was transplanted onto NSG-A2 recipient mice. Following complete wound healing, PBMC transplantation was performed.

No rejection reactions were observed before or after PBMC transplantation, and the mice did not exhibit any significant weight loss, indicating the absence of graft-versus-host disease (GVHD).

At 2 months postoperatively, epithelial hyperplasia in NSG-A2-K14E7 was significantly greater than in NSG-A2-B6.

Both the spleen and the skin contain abundant immune cells expressing hCD45+; immunofluorescence analysis reveals that the majority of these are infiltrating T cells (CD3+).

Testing a therapeutic HPV vaccine

Only the NSG‑A2_K14E7 graft exhibited a rapid response to both the first and second HPV vaccinations, with a significant reduction in the initial graft area.

When assessed ex vivo with the E7 peptide, IFNγ expression was observed only in CD4+ and CD8+ human T cells isolated from the spleens of animals vaccinated with the HPV vaccine.

Conclusion:

The F1 generation derived from crossing K14E7 transgenic mice with NSG-A2 mice exhibits characteristic epidermal thickening. When skin from the F1 mice is transplanted into NSG-A2 recipients and human PBMCs are subsequently infused, a humanized mouse skin transplantation model with human immune cell engraftment can be established.

Following vaccination, the initial tumor area of NSG-A2_K14E7 xenografts was significantly reduced. Spleen tissues harvested after inoculation were re‑immunized with the E7 peptide; IFN‑γ expression was detected only in human T cells from the NSG-A2_K14E7–vaccinated group, demonstrating that the human immune system can mount an antigen‑specific response upon administration of the anti‑tumor vaccine.

This model can effectively evaluate the response of a humanized mouse skin transplantation model to a DNA‑HPV therapeutic vaccine.

Result 3: PBMCs treated with Flt3‑L enhance immune responses in PBMC‑hu‑NSG‑A2 mice, increasing the number of human CD141+ cDC1 cells and boosting antigen‑specific T cell responses.

In vitro incubation of PBMCs with Flt3‑L increased the DC cell population at 24 h, but a decline was observed at 48 h; therefore, a 24‑hour treatment was selected for subsequent experiments.

Analysis conducted at the endpoint (Day 17) demonstrated that the detectable fraction of human CD141+ dendritic cells was capable of internalizing F‑actin‑TNE and was activated in an NY‑ESO‑1 epitope‑specific manner.

d21 harvested spleens from humanized mice and subjected the total splenocytes, which contained human CD45+ lymphocytes, to in vitro restimulation with NY-ESO-1 epitopes. The vaccine combined with FLT3L exhibited a stronger effect, resulting in a significantly higher number of epitope-specific CD8+ T cells expressing one or both cytokines.

After multiple attempts using both low- and high-dose injections of PBMCs, we found that neither regimen resulted in weight loss, possibly due to a delayed onset of GvHD. Notably, 2 × 10^6 PBMCs demonstrated significantly higher engraftment efficiency among CD45+ cells.

Conclusion: The development of therapeutic vaccines requires an adequate supply of dendritic cells.

01 In vitro incubation of PBMCs with Flt3‑L for 24 hours increased the proportion of DCs; prior to subsequent PBMC transplantation, the cells were further incubated with Flt3‑L for 24 hours.

02 The model was inoculated with a fluorescently labeled nanoemulsion containing a specific peptide, which significantly enhanced the expression of CD141+ dendritic cells; CD86+ CD141+ cDC1 cells were specifically activated by the NY-ESO epitope.

03 Following vaccination with CleC9A-TNE, splenocytes were isolated and subjected to in vitro NY-ESO-1 stimulation, revealing a significant increase in the number of epitope-specific CD8+ T cells secreting one or two cytokines.

04 2×10^6 PBMCs exhibited significantly higher engraftment efficiency, and subsequent experiments were conducted using 2×10^6 PBMCs for transplantation.

Result 4: A direct comparison of the PBMC-hu-NSG and PBMC-hu-NSG-A2 models supports the use of HLA-matched systems for testing cancer vaccines.

Differences in Two Types of Mouse Immune Cells Following Vaccination

The overall magnitude of epitope-specific T-cell responses induced in the two mouse strains was similar.

A slightly but significantly higher number of monofunctional CD8+ T cell responses was observed in PBMC-hu-NSG-A2 mice.

The effects of two mouse strains on MDA-MB-231 following vaccination

MDA-MB-231–bearing hu‑NSG mice that were unvaccinated (control) and vaccinated (TNE) began to exhibit signs of GvHD on day 24 after PBMC injection. In contrast, PBMC‑hu‑NSG‑A2 mice showed no evidence of GvHD within six weeks following PBMC engraftment. Moreover, over a 30‑day period without any observable GvHD, vaccination in PBMC‑hu‑NSG‑A2 mice resulted in significant inhibition of MDA‑MB‑231 tumor growth.

The effect of vaccination on melanoma A375 in mice

As shown in the figure, vaccination significantly inhibits tumor growth, with no evidence of GvHD observed up to day 40. In contrast, mice that did not receive the vaccine all maintained high levels of CD45+ cells.

On day 40, the spleen was harvested, and after 6 hours of in vitro stimulation with a tumor-associated epitope library followed by intracellular cytokine staining, cytokine production in splenic CD8+ and CD4+ T cells was assessed by flow cytometry.

Conclusion:

Following PBMC transplantation and vaccination in NSG and NSG‑A2 mice, T‑cell responses were broadly comparable, with CD8+ T‑cell responses slightly stronger in the PBMC‑hu‑NSG‑A2 model. After inoculation of MDA‑MB‑231 cells, GvHD developed in NSG mice by day 24, whereas in PBMC‑hu‑NSG‑A2 mice, vaccination markedly suppressed MDA‑MB‑231 tumor growth. Furthermore, in PBMC‑hu‑NSG‑A2 mice, vaccination significantly inhibited A375 tumor growth, and in vitro assays confirmed enhanced lymphocyte effector functions and robust cytokine production.

Summary

Despite ongoing progress, selecting the optimal antigenic formulation for human cancer vaccines remains a challenge, necessitating appropriate preclinical models to investigate the immunogenicity of novel human tumor antigens and to functionally characterize the CD4+ and CD8+ T-cell responses elicited in vivo. These models provide a valuable platform for evaluating the efficacy of therapeutic cancer vaccines that target HPV16‑associated dysplastic skin lesions and deliver multiple tumor antigens specifically to CD141+ dendritic cells.

Disease Model | Construction of an Alcohol-Induced Liver Injury (ALD) model, with comprehensive assessments including histopathology, biochemical analyses, Oil Red O staining, and H&E staining!

Construction of Tumor Animal Models: Colorectal Cancer Edition