by Aliyah Weinstein, PhD | Published: November 14th, 2024
Key Takeaways
Lymphodepleted mouse models play an important role in characterizing the immune cells required for particular types of immune responses, such as in the context of disease pathogenesis, vaccination, immuno-oncology, and immunotherapy. Models of lymphodepletion include genetic lymphodepletion using commercially available mouse strains such as Rag2 Knockout (KO) and B cell-deficient strains, as well as non-genetic methods including antibody-mediated depletion, chemotherapy, and radiation. Each method has unique use cases and translational relevance.
Both chemotherapy and radiation are used clinically and preclinically for lymphodepletion1. Chemotherapy or total body irradiation at low doses are referred to as non-myeloablative regimens, as they lead to reversible myelosuppression but do target peripheral lymphoid cells, for example, to eliminate host T cells in the context of CAR-T therapy2. In the preclinical setting, immuno-oncology studies have demonstrated that lymphodepletion prior to adoptive T cell transfer and/or dendritic cell-based vaccination can lead to the rejection or stabilization of an established murine melanoma3–5. Myeloablative doses of chemotherapy or radiation are also used in the context of immunotherapy; this is used when whole bone marrow or hematopoietic stem cell transplant is the interventional therapy. Chemotherapy and radiation may be the most translationally relevant methods of lymphodepletion (or myeloablation) because they are both being used clinically in immuno-oncology and transplant settings. However, their lack of specificity and potential harsh effects can be experimental concerns.
The use of antibody-based lymphodepletion allows for temporal control of lymphodepletion, as well as for normal immune development in the mice prior to the lymphodepletion step. A major advantage of this method over genetic lymphodepletion is that it can be used with any background strain of mouse. Genetically engineered and immunodeficient mouse models are commonly used in immuno-oncology studies, so the ability to lymphodeplete on top of other targeted mutations can provide robust data in this field. For example, a recent publication from Afkhami et al. (discussed in more depth below) used antibody-based depletion of T cells in B cell-deficient Jh mice to temporally control T cell depletion following vaccination for SARS-CoV-26.
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There is translational relevance of antibody-based lymphodepletion, as well. There are many antibody-based lymphodepletion schemas in preclinical or early phase clinical trials, largely in the area of transplant biology and hematopoietic stem cell transplant7. For clinical immuno-oncology applications, trials have been ongoing to evaluate the use of antibody-based lymphodepletion over traditional chemotherapy/radiation leading up to adoptive T cell transfer8. Recent preclinical studies have evaluated the impact of radiolabeled anti-CD45 antibodies as a targeted alternative to total body irradiation, also as preparation for adoptive T cell therapy or CAR-T therapy9. Therefore, antibody-based lymphodepletion is not just an experimental intervention but also part of potential therapeutic approaches.
Rag2-deficient mice contain a mutation in the Rag2 gene that inactivates the production of the Rag2 protein – a key enzyme involved in V(D)J recombination. Because of the disruption in this process, mature B and T cells cannot form in these mice. Rag2-deficient animals are available on both the B6 and BALB/c backgrounds, making these mice versatile tools for a variety of immunologic studies.
Rag2 knockout (BALB/c) mice were used in a recent study to understand the immunosuppressive role of iNKT cells in arthritis, which is a disease driven by CD4+ T cells10. The Rag2 knockout background allowed for the adoptive transfer of autoreactive T cells in this model. Using a model of psoriatic arthritis induced by mannan injection, autoreactive CD4+ T cells were adoptively transferred into the Rag2 knockout mice with or without co-injection of iNKT cells. This tightly controlled system was used to demonstrate the specific immunosuppressive effect of iNKT cells on these autoreactive CD4+ T cells, providing more specific data than performing this study in an animal with a fully competent immune system.
Several studies have also used Rag2-deficient mice to study the influence of innate lymphoid cells (ILCs) versus lymphoid cells in gut barrier damage11,12. Rag2 deficiency stalls the development of T and B cells, but not ILCs. One study looked at specific bacterial populations in the gut microbiome that protect against barrier damage, and which immune cells are required for this protection. The E. coli isolate GDAR2-2 drives ILC3 expansion in the gut and protects against C. rodentium infection and colitis in Rag2-deficient (B6) animals, suggesting that this protection is driven by ILC3s.12 In another study, to test the hypothesis that IL-33 drives ILC-mediated protection against amebic colitis, Rag2 knockout (B6) mice were treated with recombinant hIL-33 followed by amebic challenge, and mice were still protected from colitis. When the Rag2/IL2rg double KO model, which is deficient T cells, B cells, NK cells, and ILCs, was used, protection was lost.11
Indeed, Taconic’s Rag2/IL2rg double knockout model is useful for elucidating not just the influence of ILCs, but also of NK cells, when compared to the Rag2 knockout. One study used the biologic differences between these two strains to show that host NK cells were required to completely protect MB49 bladder carcinoma-bearing mice from death in conjunction with adoptive cell transfer13.
Of note, many older studies use the scid mouse, which is also deficient in T and B cells. However, compared to the Rag2-deficient models, the scid mutation is “leaky,” meaning that some functional T and B cells will in fact develop in mice bearing this mutation. Another key limitation of the scid models is their radio- and chemo-sensitivity due to the role of Prkdc in DNA damage repair.
Jh mice are also available on both the B6 and BALB/c backgrounds. These mice contain a deletion of all four JH gene segments in the Ig heavy chain locus. Because of this deletion, B cell development in these mice is drastically altered and the mice do not present with any IgM or IgG antibodies in the serum. These mice do exhibit normal T cell differentiation, and therefore this model can be used to distinguish between the influence of these two lymphoid subpopulations.
Your experimental goals will determine the best model of lymphodepletion for you to use. In a recent study evaluating the efficacy and mechanism of action of a novel COVID-19 vaccine, Afkhami et al. showed that vaccinated Jh (BALB/c) mice did not clear mouse-adapted SARS-CoV-2 from the lung compared to lymphoreplete animals6. This group also used a T cell antibody-depleting cocktail in this study, which was used to demonstrate the relative impact of B vs. T cells at different time points following vaccination. The combination of genetic and antibody-based approaches allowed the researchers to elucidate both the initiation of the vaccine response and the interaction between B and T cells. Using a Rag2-deficient animal genetically deficient in both B and T cells would not have allowed for the nuanced discoveries described in this publication.
There are many ways to lymphodeplete an animal in preparation for your preclinical study. First, determining the questions that you hope to answer with your study will inform which method(s) should be selected as well as which background strain should be used. Not discussed here are super immunodeficient mice such as those in Taconic’s NOG portfolio. These mice not only lack lymphocytes but also have deficiencies in innate immune cells such as dendritic cells and macrophages and therefore go beyond the scope of lymphodepletion discussed herein.
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