Shifting Paradigms: T Cells, FlipFlops, and Reversed T Cells

You must have come across the fundamental concepts of T cell development during your immunology class. Briefly, in the thymus, T cells expressing both CD4 and CD8 coreceptors interact with MHC molecules on thymic epithelial cells to make lineage fate decisions. CD4+ cells interact with MHCII and are called T helper cells while CD8+ cells interact with MHCI and are called cytotoxic T cells. Debates over the nitty-gritty of how this selection takes place have consisted of two main theories: “Strength of Signal model” and “Kinetic Signaling model”.

The “Strength of Signal model”, as the name suggests, focuses on the strength of the signal originating from T cell receptor (TCR) – coreceptor interactions with MHC molecules. A strong CD4-TCR signal gives rise to helper lineage while a weak CD8-TCR signal gives rise to cytotoxic lineage. According to the “Kinetic Signaling model”, the lineage decisions arise from the duration of the interaction rather than the strength. A longer/sustained TCR interaction induces the transcription factor (TF) ThPOK which paves the way to T helper cell lineage while a shorter TCR interaction induces the expression of another transcription factor called Runx3, which leads to cytotoxic T cell lineage.

On the Shifting Paradigm

From the latest study published in Nature Immunology, this debate can finally be put to rest. And spoiler alert: The Kinetic Signaling Model wins!

Shinzawa & colleagues created mice with a reversed T cell immune system, by making the CD4 gene loci code for CD8 coreceptor protein and CD8 gene loci to code for CD4 coreceptor protein. They named these mouse lines, FlipFlop.

To everyone’s surprise (and a show of nature’s staggering robustness), these Flipflop mice were perfectly healthy with almost the same immune system, with one key difference from WT mice: CD4+ cells were now cytotoxic, and CD8+ cells were helper cells! The CD4+ cytotoxic cells were driven by Runx3 while CD8+ helper cells by ThPOK while maintaining characteristic gene signatures as expected in wild-type T cytotoxic and helper cells. Wait, what? How?

Conducting a wide range of experiments, the scientists found that the CD4+ and CD8+ FlipFlop T cells were still dependent on their respective MHCII and MHCI receptors. Also, although the coreceptor proteins had been reversed, the gene loci were still the same. And it is the gene loci’s cis-regulatory elements that decide which transcription factors will be activated, and which lineage would be chosen. The gene loci also regulated how much coreceptor mRNA and proteins were present in the double-positive T cells.

Normally, CD8 coreceptor expression is enhanced in WT mice. But in FlipFlop mice, CD4 coreceptor expression was higher – pointing to CD8 gene loci causing this upregulation. They also found that despite having low expression and equally lower downstream signaling activity, CD8/MHCI activation led to higher CD5 production. CD5 is a marker for strength of TCR signaling. Given the low everything of CD8 coreceptor, the only explanation for high CD5 expression could be increased time of interaction between TCR-coreceptor and MHCI –a nod in the direction of the kinetic signaling model.

To clearly conclude the paragraph above, in FlipFlop mice, despite low CD8 coreceptor presence, long duration of CD8/MHCI interaction led to activation of ThPOK TF and high CD5 expression, leading to helper cell lineage selection. Consequently, CD4+ coreceptors being coded by CD8 gene loci were present in the cell in larger amounts and hence required only a short period of TCR-coreceptor and MHCII interaction to induce Runx3 TF and cytotoxic cell lineage. Just to state the obvious, all of this was completely opposite (or should I say, flipflop) to what normally happens in WT mice.

This data ultimately shows that strong or weak signals do not make lineage fate decisions, but the amount of time spent in the interaction decides the transcription factor expression and subsequently, the lineages. Hence, the Kinetic Signaling Model rules the throne for the foreseeable future.

Still glued to the idea of FlipFlop mice and the CD4/CD8 reversal? Read further to see how this novel immune system functions!

On Reversed T Cell Immune System

With CD4 cytotoxic cells and CD8 helper cells, what happens to the regulatory T cells (Tregs)? Tregs are normally a subset of CD4 T helper cells with FOXP3+ expression. Well, in FlipFlop mice, the CD8+ Tregs were completely viable and functioning just like in WT. They also had a similar genetic signature to WT CD4+ Tregs and prevented autoimmunity robustly.

Another subtype of WT T helper cells is CD4+ T follicular helper cells (TFH cells). Unsurprisingly, TFH cells in FlipFlop mice belonged to the CD8+ gang and just like Tregs, expressed all characteristic TFH related proteins. TFH cells are antigen-specific T cells that activate antigen-specific B cells, forming germinal centers and producing IgG antibodies. In FlipFlop mice, despite TFH cells being present in numbers similar to that of WT mice, there were neither germinal centers nor any IgG production against the specific antigen. FlipFlop B cells could produce only IgM antibodies, meaning they couldn’t interact with TFH cells to undergo class switching or affinity maturation. We can blame this on the TFH cells here being CD8+ instead of CD4+, meaning they cannot interact with MHCII on the B cells.

This brings us to the why TFH antigen specificity arose in the first place: because dendritic cells which would normally activate TFH cells through MHCII, in FlipFlop mice could activate them by cross-presentation of the antigen on MHCI, something B cells are incapable of doing. Mind-blowing right?

The last question they asked was, how does the FlipFlop immune system deal with viral infections? Not well! Clearance of viral infections largely depend on CD8+ cytotoxic T cells that rapidly destroy infected cells through CD8/MHCI interactions. In FlipFlop mice where cytotoxic cells are CD4+, they seek and destroy cells presenting viral antigens through MHCII, funnily interfering with the antiviral immune response instead of the actual viral infection. Moreover, infected cells that are presenting viral antigens through MHCI are not cleared because CD4+ T cells cannot read MHCI, making overcoming viral infections nearly impossible.

Hence, the reversed T cell immune system fascinatingly functions surprisingly well but due to its susceptibility to infections, also throws light on why evolution chose to go the other way round.


Shinzawa M, Moseman EA, Gossa S, Mano Y, Bhattacharya A, Guinter T, Alag A, Chen X, Cam M, McGavern DB, Erman B, Singer A. Reversal of the T cell immune system reveals the molecular basis for T cell lineage fate determination in the thymus. Nat Immunol. 2022 May;23(5):731-742. doi: 10.1038/s41590-022-01187-1.

Article author: Kevin Merchant. Kevin is a MS student at LMU Munich, Germany, who is passionate about Immunology and writing. He aims to simplify latest research so that it becomes accessible to all.

Editor: Sutonuka Bhar. Sutonuka is a scientist working on biotherapeutics development. She achieved her doctoral degree from University of Florida where her work focused on host immune responses against viruses and bacterial membrane vesicles.

Check out Antibuddies’ blog post “Shifting Paradigms: T Cells, FlipFlops, and Reversed T Cells”.





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