Dr. Courtney Ferrebee recently presented a
webinar titled "Applications of Transgenic HLA Mice, From Vaccines to Immuno-oncology". The webinar presented an overview of Taconic Biosciences' catalog of
class I and class II transgenic HLA models, including an introduction to the HLA system and the role it plays in therapeutic applications, as well as the history of how Taconic's models were generated. She then presented various specific case studies and applications for these models in vaccine and infectious disease research, immuno-oncology, and autoimmune disease research. Finally, she discussed experimental considerations, limitations, and challenges when using HLA transgenic mice to model human health and disease in preclinical studies.
Following her presentation, Dr. Ferrebee addressed many questions from webinar participants. Here we present the full webinar Q&A.
Q: There are so many in silico tools to predict HLA binding now. How do transgenic HLA mice and in silico tools fit together in vaccine development?
Dr. Courtney Ferrebee: Great question. This goes back to the many studies in the presentation that highlighted epitope identification and immunogenicity testing using the HLA transgenic mice. Using these models as tools to test and answer these questions opens a path (window) of discovery for researchers in many therapeutic areas.
Q: Are there mouse models for other HLA alleles besides A, B, and DR (or D in general) subtypes?
CF: A wide variety of transgenic HLA mouse models have been generated over the years, some of which may be available from other vendors or repositories. However, those available at Taconic carry the
A1 (CB6F1),
A2.1 (CB6F1),
A11 (CB6F1),
A24 (CB6F1),
B7 (C;B6),
B44 (CB6F1),
DR4 alleles.
Q: Are the other HLA alleles and pseudogenes or alleles relevant to human health and disease?
CF: Other HLA alleles relevant to human health and disease not discussed include:
- HLA-A26: Leukemia
- HLA-B27: Reactive Arthritis, HIV/AIDS Progression
- HLA-DR2: SLE Lupus, Multiple Sclerosis
- HLA-DR3: Hepatitis
Most of the HLA pseudogenes are not functional. However, for example, HLA-H has some transcriptional activity and recent studies have shown that it can have some functional relevance. I think more studies need to be performed to determine the biological significance of HLA pseudogenes.
Q: Does Taconic provide pure B6 background mice for these models? Why are they provided on a mixed background?
CF: Taconic's HLA Tg mice are maintained as foundation colonies on a B6 background — these are available in limited quantities. The saleable product for most of our HLA Tg lines is generated by breeding HLA Tg males with wildtype BALB/c females to produce transgenic F1 hybrids. These CB6F1 hybrids are genetically identical and yet heterozygous for all autosomal loci that differ between C57BL/6 and BALB/c genomes and thus lack many of the inherent idiosyncrasies of either parental line. One benefit of this is a principle known as hybrid vigor, in which F1 hybrids are heartier with fewer health issues compared to either inbred parental line. A second advantage is that the immune responses of CB6F1 hybrids are often intermediate compared to the skewed immune responses that differ between C57BL/6 or BALB/c genetic backgrounds. According to Dr. Sette, one of the original inventors of these mice, this F1 background improves the spectrum of detectable HLA-restricted immune responses. Taconic has not independently confirmed this conclusion.
Q: How do I differentiate between human HLA and Mouse MHC restricted responses?
CF: This is challenging due to the lack of human cytokines and human immune cells as a readout in these animals. Since HLA transgenic mice co-express murine molecules, it is required to demonstrate that T cells recognize an antigen-presenting cell (APC) that expresses the HLA transgene and do not respond to an APC that does not. In such cases, dual restriction would apply if both were recognized. As a starting point, you should determine whether your peptide binds to human HLA and murine MHC similarly and determine if similar downstream responses are seen.
Q: Can I perform syngeneic tumor engraftments in the F1 background HLA mice? If yes, what background strain should I select for my tumor?
CF: Yes. Because CB6F1 mice possess equal genetic contributions from each of the B6 and BALB/c parental strains, syngeneic tumor cell lines from either strain should successfully engraft, in most cases. For example, MC-38 CDX, which is on the B6 background, or the BCL1 CDX, which is on a BALB/c background, can be engrafted onto a CB6F1 due to the hybrid nature of the F1 background.
Q: Are you aware of any sex differences in the HLA Tg models? Should I use both male and female mice for my studies?
CF: Not aware of any sex difference in the class I transgenic models. But for class II transgenics and associated autoimmune diseases like arthritis, in HLA-DR4 male disease phenotype is more severe. Encourage pilots/collaborations.
Q: Have you seen any change in demand in the transgenic HLA mice?
CF: The various HLA mice offered by Taconic continue to show high demand due to a current uptick in interest which appears to be correlated with increasing usage in immuno-oncology studies as well as applications related to COVID-19 vaccine research.
Q: Are there age-dependent HLA allele-related diseases?
CF: This is a great question and a very interesting theory across a number of age-associated diseases. HLA-DR and DQ alleles could influence the early onset of T1D and arthritis and certain populations. I think this point highlights the potential for age-dependent, or more specifically, age-associated disease related to allelic presence in the population being a factor. It is also important to note that certain HLA alleles may have different roles within the different periods of the lifespan
1. It would be interesting to investigate this in our HLA-DR4 mice and we would be open to collaborations interested in looking at age-related (and sex-related) differences in arthritis studies.
Q: Do you have recommendations for validation of immunodominance above IFNg expression through CTL stimulation?
CF: Immunodominance is strongly correlated with HLA binding and IFNg-based CTL responses. Immunogenicity testing is one of the best approaches to determining immunodominance between HLA and MHC. Other assays to determine immunodominance include functional readout assays to determine cytotoxic T cell activation such as the Cr-51 assay and assessing T cell proliferation. You may also analyze other downstream pathways for post Th1/Th2 responses such as cytokine expression (e.g., IL2) and IgG production.
Q: We are targeting tumor-associated macrophages. Have there been successful use of this mouse line for cancer study?
CF: This is a great question. Because HLA expression is important for antigen presentation on macrophages, the presence of HLA would influence tumor-associated macrophages (TAMs). It has been established that HLA expression can play a role in cancer and define the cellular phenotype in the tumor microenvironment (TME). Tumor cells and TAMs can exploit MHC class I and II molecules to modulate cells, and specifically immune cells, in the TME. HLA molecules can downregulate macrophage activation through inhibitory factors and regulate NK cell and CD8 function as well. This could create more of an anti-inflammatory environment vs. pro-inflammatory, triggering an M2 polarization that can lead to immune cell escape/evasion and affect overall tumor development. Unfortunately, we do not have information in Taconic's models assessing HLA expression and TAMs
2. Other IO-based studies have shown demonstrated that you can use HLA Tg mice to investigate APC presentation in HLA matched tumors for immunotherapy
3,4. We would love to collaborate with your research group to generate TAM IO data. Please
reach out to us concerning this.
Q: What promoter is used to drive expression of the HLA-H2 chimeric transgene for the HLA class I alleles?
CF: Murine H2-Kb promoter.
Q: Have there been broad assessments of tissue expression for the class I HLA transgenic mouse lines (are these transgenes expressed only in the lymphocytes or are they expressed in most mouse tissues)?
CF: Transgene expression for HLA I class molecules is predominantly on white blood cells in the spleen, and this has been shown through FACs validation of the parental B6 line lymphocytes. We also have heard from customers that there is expression of HLA-A2.1 on T cells, B cells, and monocytes in the 9659 line. This has also been established in published papers.) It is also expressed in other nucleated cells for native class I MHCs but not much is known about this. Based on our discussions with the researchers who generated the animals, expression in other tissues would include the liver, and this is quite low. We do not have any additional data concerning the full extent of tissue expression of HLA class I molecules in the transgenic mice. Based on what we know concerning class II transgenics (HLA-DR) and their expression patterns, we would expect there to be differential expression throughout tissues in the animals
5.
Q: Have groups used the transgenic HLA mice to conduct unbiased antigenic epitope identification by immunizing with full-length protein and allowing the mice to process and present peptides on the transgenic HLAs?
CF: Data showing a CTL response with immunization with whole protein varies and I think this is based on the protein itself. One such study showed a CTL response to the positive control in the experiments, TLA or T. gondii lysate antigen, which is the full-length protein to smaller HLA-A2 peptide epitopes. HLA-restricted experiments in HLA-A2+ human cells did not show a response
6, suggesting that HLA restriction is not a factor in this. TAP as well as other components of the antigen presenting process are still functional. Other studies have shown responses to decaepitopes suggesting that the size of the peptide epitope is not an issue
7.
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References:
1. Caruso, C. et al. HLA, aging, and longevity: a critical reappraisal. Hum Immunol 61, 942-949 (2000).
2. Marchesi, M. et al. HLA-dependent tumour development: a role for tumour associate macrophages? J Transl Med 11, 247 (2013).
3. Koya, R. C. et al. Kinetic phases of distribution and tumor targeting by T cell receptor engineered lymphocytes inducing robust antitumor responses. Proc National Acad Sci 107, 14286-14291 (2010).
4. Wang, Q. J. et al. Identification of T-cell Receptors Targeting KRAS-Mutated Human Tumors. Cancer Immunol Res 4, 204-214 (2016).
5. Giacomini, P. et al. Tissue-specific expression of the HLA-DRA gene in transgenic mice. Immunogenetics 35, 425-425 (1992).
6. Cong, H. et al. Towards an immunosense vaccine to prevent toxoplasmosis: Protective Toxoplasma gondii epitopes restricted by HLA-A*0201. Vaccine 29, 754-762 (2011).
7. Alexander, J. et al. A Decaepitope Polypeptide Primes for Multiple CD8+ IFN-γ and Th Lymphocyte Responses: Evaluation of Multiepitope Polypeptides as a Mode for Vaccine Delivery. J Immunol 168, 6189-6198 (2002).
