T Cell Cytolytic Capacity is Independent of Initial Stimulation Strength

Original Article: PDF T cell cytolytic capacity is independent of initial stimulation strength

Cytotoxic T-cells, also referred to as cytotoxic lymphocytes (CTLs) are important for the immune systems response to viral pathogens and tumors. CTLs fulfill this role by killing infected cells or tumor cells which is done via degranulation. CTLs contain granules filled with compounds that once released from the cell (degranulation) kill the nearby targeted infected or tumor cell. Before CTLs can complete perform this selective killing they must first be activated to express the necessary molecular machinery to produce and release granules. To become activated CTLs must interact with a ligand, this ligand is an antigen bound to a molecule called MHC class 1, on another cell^2,3. MHC class 1 presents proteins from inside the cell on the cell surface. When a cell is infected MHC class 1 can present some of the proteins from the virus on the cell surface. Before CTLs are activated they are referred to as naïve CD8⁺ cells2,3. When the unactivated (naïve) CD8⁺ cells receptors bind to an MHC class 1 that is presenting a protein that shows that the cell is infected, a signaling pathway is started. This signaling pathway then leads to the production of the genes needed to activate the CTL so that it can destroy the infected or tumor cell.  The question of how the potency of the ligand, how strong of a response it elicits, affects the activation of the CTLs was the topic of this article. Other researchers have shown that in areas of the body other than lymphoid tissue, stronger ligands produce a larger T-cell response^4,5,6,7,8,9. They determined that rather than altering the route of activation within the cell or the cells phenotype after activation, potency determines how rapidly and simultaneously the cells initiated activation¹.

To determine how ligand potency affects CTL activation the researchers first needed to determine the phases of the process. This was done by initiating activation in CTLs and characterizing the transcriptional changes through single cell RNA sequencing during initial 6 hours of cell activation¹. By sequencing the mRNA being produced at different time intervals during activation they could distinguish different genes that were activated at these different intervals, thus showing distinct phases through transcriptional changes. They identified 2 phases of the activation response, early activated and late activated.

From this point the researchers moved forward to study ligand potency on specifically the early activation phase. They selected two genes characteristic of this phase and used flow cytometry to asses expression while modulating the potency of ligand¹. It was found that the higher potency ligands induced increased expression quickly that then decreased later in the 6 hours; whereas the lower potency ligands showed a delayed increase in expression. The same levels of these transcription factors were induced just at different times in the 6-hour activation period. This shows that in this case that ligand potency determines the rate of the response not the pathway of the response itself. In other terms this shows that the stimulation strength (ligand potency) controls the probability that a cell could activate, regulating the rate the pathway of activation is initiated, not how fast it occurs inside the cell once it has begun.

Fig. 2: Early-response genes can be TCR dependent or TCR independent.

The researchers also needed to test if the potency of ligand affected the type of effector cell the naïve CD8⁺ cells would differentiate into once activated. Effector cells are cells that carry out designated functions. To determine if ligand potency affected what effector cells would arise the researchers used mass cytometry to measure amounts of proteins related to differentiation and effector function¹. No largely significant differences were found between cells that had been activated by high potency ligand and those that had been activated by lower potency ligands. All cells had the same probability values for differentiating into the different effector cell types. This shows that ligand potency does not have a hand in determining the effector function of the CTLs.

Lastly the researchers needed to determine if ligand potency affected the differentiated cells cytolytic ability. Cytolytic ability is the cells ability to degranulate and kill the targeted cell. The researchers identified the protein LAMP1 that is present in the granules. During degranulation LAMP1 is trafficked to the cell membrane to be released¹. Thus, by tracking the quantity of LAMP1 that is trafficked to the cell membrane the cells cytolytic ability can be determined. This experiment showed that all the activated cells demonstrated cytolytic ability regardless of whether they were activated with high or low potency ligand.

Through these experiments the researchers showed that the ligand potency (signal strength) determines how rapidly and uniformly a population of naïve CD8⁺ cells will activate. This information sheds light on how this aspect of the immune response is controlled. These findings could potentially contribute to research concerning tumor suppression and treatment. Knowing that the potency of the ligand does not affect the mechanism of activation but rather how rapidly it occurs in a population of CTLs could aid in narrowing down the focus of research into treatments using CTLs to target tumors. This research also points us in a direction of where to go next. Now that we know ligand potency doesn’t affect the type of effector cells that naïve CD8⁺ cells differentiate into nor their cytolytic ability, we can investigate other factors that might. By doing this, if we find answers to these questions, we could design treatments to reduce cytolytic activity in CTLs when there is a malfunction that causes their overactivity in a patient, along with treatments for other illnesses that involve CTLs.

References

1. Richard, A. C. et al. T Cell Cytolytic Capacity is Independent of Initial Stimulation Strength. Nature Immunology. 19, 849-858 (2018).

2. Brownlie, R. J. & Zamoyska, R. T cell receptor signalling networks: branched, diversified and bounded. Nat. Rev. Immunol. 13, 257–269 (2013).

3. Cantrell, D. Signaling in lymphocyte activation. Cold Spring Harb. Perspect. Biol 7, a018788 (2015).

4. Ozga, A. J. et al. pMHC affinity controls duration of CD8+ T cell-DC interactions and imprints timing of effector differentiation versus expansion. J. Exp. Med. 213, 2811–2829 (2016).

5. Zehn, D., Lee, S. Y. & Bevan, M. J. Complete but curtailed T-cell response to very low-affinity antigen. Nature 458, 211–214 (2009).

6. Skokos, D. et al. Peptide-MHC potency governs dynamic interactions between T cells and dendritic cells in lymph nodes. Nat. Immunol. 8, 835–844 (2007).

7. Denton, A. E. et al. Affinity thresholds for naive CD8+ CTL activation by peptides and engineered influenza A viruses. J. Immunol. 187, 5733–5744 (2011).

8. King, C. G. et al. T cell affinity regulates asymmetric division, effector cell differentiation, and tissue pathology. Immunity 37, 709–720 (2012).

9. Palmer, E., Drobek, A. & Stepanek, O. Opposing effects of actin signaling and LFA-1 on establishing the affinity threshold for inducing effector T-cell responses in mice. Eur. J. Immunol. 46, 1887–1901 (2016).

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