🦠 microbiology

Immune receptor LILRB1 mediates cis-signalling which is targeted by RIFINs of the malaria parasite

This study reveals that *Plasmodium falciparum* RIFINs exploit the dynamic conformational equilibrium of the immune receptor LILRB1—stabilizing either an elongated form for trans-signalling or a buckled form for cis-signalling via MHC class I—to suppress host immune responses and ensure parasite survival.

Original authors: Chamberlain, S. G., Widdess, M., Morch, A., Sakoguchi, A., Sakuno, R., Kurz, E., Chen, L., Valvo, S., Iwanaga, S., Dustin, M., Higgins, M. K.

Published 2026-04-17

📖 5 min read🧠 Deep dive

CC BY 4.0

Original authors: Chamberlain, S. G., Widdess, M., Morch, A., Sakoguchi, A., Sakuno, R., Kurz, E., Chen, L., Valvo, S., Iwanaga, S., Dustin, M., Higgins, M. K.

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

The Big Picture: A High-Stakes Game of Hide-and-Seek

Imagine your body is a fortress, and the immune system is the army of guards (like Natural Killer cells) patrolling the walls. Their job is to spot invaders and destroy them.

The malaria parasite (Plasmodium falciparum) is a master spy. It hides inside your red blood cells, but it knows the guards are coming. To survive, the parasite has evolved a massive arsenal of "disguises" called RIFINs. These are proteins that stick out of the infected blood cell like little flags.

The parasite's goal? To trick the immune guards into thinking, "Oh, this is a friendly cell. Don't shoot!"

The Old Story: One Trick, One Lock

For a while, scientists knew the parasite used one specific trick. The parasite's RIFINs would grab onto a specific "Do Not Kill" button on the immune guards called LILRB1.

Think of LILRB1 as a smart lock on the guard's door.

Normally, the guard's own ID card (MHC Class I) fits into this lock to say, "I am one of you, stand down."
The parasite's RIFINs were known to mimic that ID card. They would grab the lock in a specific way (an "elongated" shape), tricking the guard into lowering their weapon.

The New Discovery: Two Tricks, Two Locks?

This new paper reveals that the parasite is much smarter than we thought. It doesn't just have one trick; it has two different groups of RIFINs that use two different strategies to jam that same lock.

Strategy 1: The "Long Reach" (The Elongated Shape)

Some RIFINs grab the lock from a distance, keeping the lock straight and stretched out. This is the "trans" signal. It's like a spy reaching across a table to press the "stop" button on a guard standing on the other side of the room.

Strategy 2: The "Buckle" (The C-Shaped Twist)

Here is the big surprise. The researchers found a second group of RIFINs that don't just grab the lock; they bend it.

Imagine the lock is a long, flexible ruler.

The first group of RIFINs grabs the ruler while it's straight.
The second group grabs the ruler and buckles it into a "C" shape.

Why does bending the lock matter?
When the lock is bent into a "C" shape, it suddenly fits perfectly with a different part of the guard's own body—specifically, the guard's own ID card (MHC Class I) that is sitting right next to it on the same cell.

This creates a "Cis" signal. Instead of reaching across the room to press the button, the bent lock grabs the guard's own ID card and forces them to hug it. This internal hug sends a powerful "Stand Down" signal right inside the guard's own cell.

The "Buckled" Breakthrough

The scientists proved this by:

Taking a 3D photo (Cryo-EM/X-ray): They saw the lock bent into a "C" shape when the second group of RIFINs grabbed it.
Building a model: They created a "locked" version of the receptor that was permanently bent. They found that this bent version was better at grabbing the guard's own ID card and shutting down the immune response.
Testing the guards: When they put these bent locks on immune cells, the cells stopped attacking. Even better, when they added the second group of RIFINs, the cells stopped attacking even more effectively.

Why Does the Parasite Need Two Tricks?

You might ask, "Why evolve two different ways to do the same thing?"

Think of the immune system as a thermostat.

Sometimes the "Do Not Kill" button is easy to reach (straight lock).
Sometimes the environment makes it hard to reach (bent lock).

By having two groups of RIFINs, the parasite ensures that no matter how the immune guard is positioned or how its internal "thermostat" is set, the parasite can always find a way to jam the signal.

If the guard is in a "straight" mode, the first RIFIN group works.
If the guard is in a "bent" mode (perhaps due to high levels of its own ID cards nearby), the second RIFIN group works.

It's like a burglar who has both a skeleton key (for straight locks) and a lock-picking tool (for bent locks). No matter what kind of lock the house has, the burglar gets in.

The Takeaway

This paper solves a mystery about how malaria survives. It shows that the parasite is a master of disguise with a dual strategy:

Trick the guard from the outside (Trans signaling).
Force the guard to hug its own ID card (Cis signaling) by bending the receptor.

This "buckling" mechanism is a new way the parasite suppresses our immune system. Understanding this gives scientists a new target for vaccines or drugs. If we can figure out how to stop the parasite from bending the lock, or how to force the lock back to its straight shape, we might be able to wake up the immune guards and let them destroy the malaria parasite.

1. Problem Statement

Context: Plasmodium falciparum, the deadliest malaria parasite, evades immune clearance by expressing surface proteins called RIFINs on infected red blood cells (iRBCs).
Known Mechanism: Previous studies established that RIFINs bind to the inhibitory immune receptor LILRB1 (Leukocyte Immunoglobulin-like Receptor Subfamily B Member 1) on Natural Killer (NK) cells and $\gamma\delta$ T cells. This binding mimics MHC class I, triggering inhibitory "trans" signaling that suppresses immune cell activation and perforin release.
The Mystery: Two distinct groups of RIFINs were identified:
1. Those binding the D2 domain of LILRB1 (overlapping the MHC class I site).
2. Those binding the D3D4 domains of LILRB1 (identified via unique antibodies in malaria-exposed individuals).
The Gap: While D2-binding RIFINs were known to stabilize an elongated LILRB1 conformation, the structural and functional consequences of D3D4-binding RIFINs were unknown. Specifically, it was unclear if LILRB1 could adopt different conformations on the immune cell surface and if these different conformations mediated distinct inhibitory mechanisms (e.g., "cis" vs. "trans" signaling).

2. Methodology

The authors employed a multidisciplinary approach combining structural biology, biophysics, cell biology, and immunology:

Structural Biology:
- X-ray Crystallography: Solved the crystal structure of full-length LILRB1 in complex with a D3-binding RIFIN (D3-RIF) and a second D3-binding RIFIN (D3-K-RIF).
- Superposition: Compared these new structures with previously known elongated LILRB1 structures and MHC class I complexes.
Biophysical Characterization:
- FRET (Förster Resonance Energy Transfer): Designed a LILRB1 FRET sensor (flanked by mCerulean and mCitrine) to monitor conformational changes (distance between N- and C-termini) in solution upon RIFIN binding.
- Surface Plasmon Resonance (SPR): Measured binding kinetics of various RIFINs to LILRB1 and antibodies.
- Biolayer Interferometry (BLI): Tested simultaneous binding of RIFINs and MHC class I to LILRB1.
Cellular & Molecular Engineering:
- Mutagenesis: Created a LILRB1BUCLD mutant by introducing a disulfide bond to "lock" LILRB1 in a buckled conformation.
- CRISPR-Cas9: Generated NK92 cells and HEK293 cells lacking $\beta2$ -microglobulin ( $\beta2M^{-/-}$ ) to eliminate endogenous MHC class I for controlled experiments.
- Library Screening: Generated transgenic P. falciparum libraries expressing individual RIFINs to screen for D3D4 binders using flow cytometry and amplicon sequencing.
Functional Assays:
- Supported Lipid Bilayers (SLB): Used SLBs coated with activating ligands (ICAM-1, CD58, B7-H6) and RIFINs to induce immune synapses. Measured perforin deposition as a readout of NK cell inhibition.
- FCCS (Fluorescence Cross-Correlation Spectroscopy): Measured the co-diffusion (cis-interaction) of LILRB1 and MHC class I on the cell surface.
- FRET Signaling Reporters: Engineered a reporter to measure ITIM phosphorylation (inhibitory signaling) in real-time.
- Structured Illumination Microscopy (SIM): Visualized LILRB1 microcluster formation.

3. Key Contributions & Results

A. Structural Discovery: The Buckled Conformation

Finding: Crystal structures revealed that D3-binding RIFINs force LILRB1 into a C-shaped (buckled) conformation.
Mechanism: The RIFIN binds a hydrophobic pocket on the D3 domain that is normally involved in D2-D3 interdomain contacts. This binding prevents the elongated state, bending the receptor so the D1 and D4 domains come into proximity.
Validation: FRET assays confirmed that D3-binding RIFINs (but not D2-binding RIFINs) induce this buckling in solution.

B. Identification of a RIFIN Clade

Finding: Screening of P. falciparum libraries (3D7 and Kenyan strains) identified a specific phylogenetic clade of RIFINs that bind the D3D4 domains.
Consistency: All members of this clade stabilize the buckled LILRB1 conformation and bind with high affinity.

C. The "Cis" Signaling Mechanism

Discovery: The buckled conformation of LILRB1 creates a surface compatible with binding MHC class I on the same cell (cis-interaction).
Evidence:
- FCCS showed that the locked buckled mutant (LILRB1BUCLD) has a significantly higher propensity to co-diffuse with MHC class I than wild-type LILRB1.
- This cis-complex formation was blocked by the anti-MHC antibody W6/32.
Signaling: The LILRB1-MHC cis-complex triggers ITIM phosphorylation and recruits SHP-1, generating a basal inhibitory signal. This was confirmed by FRET reporters and reduced perforin secretion in NK cells.

D. Functional Divergence of RIFINs

D2-Binding RIFINs: Bind the elongated LILRB1, competing with MHC class I for "trans" binding (on target cells). They inhibit NK cells via standard trans-signaling.
D3-Binding RIFINs: Bind the buckled LILRB1. Crucially, they enhance the cis-interaction between LILRB1 and MHC class I.
- BLI assays showed that MHC class I actually promotes D3-RIF binding (by stabilizing the buckled state), whereas it inhibits D2-RIF binding.
- In NK cells, D3-RIFs suppress perforin release more potently than D2-RIFs, and this suppression is dependent on the LILRB1-MHC cis-complex.

E. Dynamic Equilibrium Model

The paper proposes that LILRB1 exists in a dynamic equilibrium between elongated and buckled states.
Cis-Ligands (MHC class I on the same cell): Stabilize the buckled state, setting a high threshold for activation (basal inhibition).
Trans-Ligands (RIFINs or MHC on target cells): Shift the equilibrium toward the elongated state to trigger specific signaling.
Parasite Strategy: The parasite evolved two RIFIN clades to exploit both states:
1. D2-RIFs target the elongated form (trans).
2. D3-RIFs target the buckled form, reinforcing the cis-inhibitory signal to further dampen immune responses.

4. Significance

Novel Mechanism of Immune Evasion: This is the first experimental demonstration that cis-signaling through an inhibitory receptor (LILRB1) actively suppresses immune cell function. It challenges the view that cis-interactions merely compete with trans-ligands; here, they actively set the signaling threshold.
Conformational Plasticity: It reveals that immune receptors can adopt distinct, stable conformations (elongated vs. buckled) on the cell surface, each with different ligand specificities and signaling outcomes.
Parasite Adaptation: The study explains why P. falciparum maintains two distinct RIFIN clades. By targeting both the elongated and buckled conformations of LILRB1, the parasite ensures robust immune suppression regardless of the host cell's MHC class I density or the specific conformational state of LILRB1.
Therapeutic Implications: Understanding the "buckled" state and the cis-signaling mechanism opens new avenues for malaria vaccine design or immunotherapies that could disrupt these specific inhibitory complexes to restore NK cell activity.

Conclusion

The paper elucidates a sophisticated immune evasion strategy where P. falciparum RIFINs exploit the conformational flexibility of the host receptor LILRB1. By stabilizing a "buckled" conformation, a specific clade of RIFINs promotes a cis-interaction with MHC class I, generating a potent basal inhibitory signal that raises the activation threshold for NK cells, thereby preventing parasite clearance.