Adoptive T Cell Transfer Model
In the tumor setting the aim of immunotherapies is to mobilize the immune system and/or enhance or re-invigorate the quality of the existing immune response. In order to evaluate whether a therapeutic enhances the immune response we utilize models, outside of the disease-specific setting, where the immune response is suoptimal so this can be enhanced by a therapeutic. One such approach to evaluate modulation of T cell function is the use of the OT-I and OT-II TCR (T cell receptor) transgenic models to measure regulation of specific T cell responses in vivo in response to their cognate antigen Ovalbumin (OVA).
The OT-I mouse is transgenic for a TCR that recognizes an OVA-derived peptide SIINFEKL presented on MHCI (H-2Kb) , in this mouse nearly all T cells are CD8+ and have the same TCR. The OT-II mouse is transgenic for a TCR that recognizes the OVA-derived peptide OVA323-339 presented on MHCII (I-Ab). In this mouse nearly all T cells are CD4+ and have the same TCR. These cells can be isolated, labelled and adoptively transferred into a recipient mouse at the desired frequency. This provides a system where the frequency of T cells able to respond to a defined antigen is raised to a sufficient level where they can be tracked and evaluated ex vivo in a way that is not possible when looking at an endogenous T cell response. The adoptively transferred cells are activated by injection of either whole OVA/Alum or the relevant OVA peptide/alum, depending on the kinetics required and whether antigen processing is required as part of the model. The effect of a test therapeutic on this defined T cell population in terms of activation, effector profile and survival can then be measured both in the draining lymph nodes and distally in the spleen or systemically in the blood.
Tommaso Iannitti, Group Leader, Pharmacology for Charles River Discovery Services, discusses adoptive T-cell transfer models for screening immunotherapies.
Flow cytometry analysis ex vivo allows examination of the OT-I or OT-II population; proliferation of these cells can be tracked by examining dilution of a dye such as cell trace violet and expression of effector molecules which are required for cytotoxic function of CD8 T cells such as Granzyme B can be measured. Cytokine profiles of cells can be measured ex vivo such as IL-2, IFNg and TNFa. This can be further validated by luminex analysis of cytokines in serum. By examining surface markers on the cells, expression of your target molecule can be evaluated alongside expression of both inhibitory and co-stimulatory molecules which may influence the immune response. For the OT-II adoptive transfer system effects on T cell polarization and production of regulatory cytokines or phenotypes, such as Foxp3 expression can be evaluated.
- Cytokine profile (IL-2, IFNg)
- Effector molecule expression (Granzyme B)
- Proliferation (cell trace violet dilution)
- Surface and intracellular staining for up to 16 parameters for flow cytometric analysis panel includes congenic markers, CD4, CD8, activation markers (CD25, CD69, CD62L)
- Customized selection of co-stimulatory/inhibitory receptors
- ICC staining for cytokines intracellular markers
Figure 2: Effect of checkpoint inhibitors on CD8 T cell activation and effector function: Effector molecule and activation marker expression: OT-I CD8 T cells were adoptively transferred into recipient mice and primed via SIINFEKL/Alum injection. Draining lymph nodes were processed, stained and analyzed by flow cytometry. Data shows cells gated through single, viable, scatter gates and subsequent analysis of the OT-I population is shown.
Figure 3: Effect of checkpoint inhibitors on OT-I CD8 T cell activation and effector function: Effector molecule and activation marker expression: OT-I CD8 T cells were adoptively transferred into recipient mice and primed via SIINFEKL/Alum injection. Draining lymph nodes were processed, stained and analyzed by flow cytometry. The percentage of CD8+ OT-I T cells expressing the effector molecules IFN, granzyme B and the activation markers CD25, and CD69 were determined by flow cytometry. Each point represents an individual mouse (N=10 per group) and error bars show the mean +/- SEM. Checkpoint inhibition increases the percentage of cells expressing IFN and GranzymeB and the percentage of cells positive for CD25.
What is the purpose of Pharmacodynamic models?
Pharmacodynamic, or PD models, allow us to examine the role of a therapeutic outside of the disease model setting. And the value of this is that you get information as early as possible across multiple assay types, on whether your therapeutic is hitting its target, and whether it's impacting the desired cell type, in the manner in which it should.
What is the difference between immuno-oncology PD models and other PD models?
We can think of PD models in two different ways. If we're thinking about a therapeutic which might target a tumor, so in an immuno-oncology setting, the immune response is often low or suboptimal and T-cells are often exhausted, so in this model we sub-optimally activate our T-cells. This then allows us to see whether your therapeutic enhances the immune response and look at effector function of the T-cells, and whether they're doing what you'd expect. And therefore can predict efficacy in syngeneic models by looking at the efficacy of test compounds on key markers of T cell activation and effector function such as interferon gamma and granzyme B.
Conversely, if you're looking to develop a drug in the autoimmune setting, then what you're aiming to do is take a very strong T-cell response, because T-cells in that setting would often be improperly activated, and what we want to do, is generate a situation where we regulate, and dial down that immune response. So, what we do with the OT1 cells in this setting, is activate them in an optimal manner, and then we can look at the effect of your therapeutic to regulate that response. So, this approach adds value because it tells you early on whether your therapeutic is hitting its target, before you move through to the complex tier of the disease model.
Can I examine the effect of my therapeutic on both CD4 and CD8 T cell responses?
Where the aim is to examine the effect of the therapeutic on both CD4 and CD8 T cell responses OT-I and OT-II T cells can be co-transferred into the same recipient which provides the benefit of reducing animal numbers.
Can I use the PD model for screening?
Where wider screening and gene expression profiling is required nanostring analysis of the adoptively transferred cells can be performed to determine how a therapeutic is ¬¬influencing gene expression profile and pathways it may be regulating.
Can I use the adoptive T cell transfer model in conjunction with syngeneic models?
Where analysis of TIL (tumor infiltrating lymphocyte) populations are required as follow on experiments the same adoptive transfer model can be used into mice with syngeneic tumors expressing the OVA antigen as a model tumor antigen. This again raises the frequency of T cells responding to an antigen expressed specifically by the tumor allowing the effect of a novel therapeutic on a specific anti-tumor tumor response to be measured in the way described above.