Early resident NK cell response to local HIV infection in lymphoid tissue

Background

Human Immunodeficiency Virus or HIV is a virus that invades the body’s defenses by attacking the immune system, specifically CD4+ T cells (Kaide et al., 2021; Katz, May, Baker, & Test, 2011). HIV hijacks the replication and transcription machinery of CD4+ T cells, including enzymes and cellular structures required for viral genome replication, to multiply and spread throughout the body. HIV can integrate its genetic material into the DNA of the CD4+ T cells. As soon as the virus enters a CD4+ T cell, the viral reverse transcriptase begins to convert RNA into DNA. From here, the viral integrase catalyzes insertion of the viral DNA into the host cell’s genome. This allows the virus to become active and begin to make new virus particles or go into a latent state, where it no longer produces new virus particles.

HIV tends to seek residency in the lymphoid tissue, such as the lymph nodes and tonsils, where CD4+ T cells are very abundant. Once HIV infiltrates these sites, it can establish latency by hiding in long-lived cells known as viral reservoirs. Despite treatment, HIV can persist in these reservoirs, where it remains dormant and evades immune detection, preventing complete eradication. This stage is referred to as Acquired Immunodeficiency Syndrome or AIDS. Since the virus can transition back and forth between the dormant and active state, it makes it nearly impossible for antiretroviral therapy to recognize and fully eliminate the virus.  Current antiretroviral therapies suppress the viral load and improve the duration and quality of life of people living with HIV. However, these antiretroviral therapies are only successful at suppressing the viral load, requiring lifelong administration to prolong the life expectancy of HIV patients.

As our understanding of both HIV and human biology increases, new approaches are being formed to deal with the therapeutic challenges HIV presents. Currently, an active area of interest is the role of Natural Killer (NK) cells in HIV infection. NK cells are cytotoxic innate lymphoid cells which can serve innate or adaptive-like functions to target and destroy infected or otherwise stressed cells in the body (Mace, 2023). Characteristically, NK cells have the ability to target and destroy cells without needing prior antigen presentation (Lanier et al., 1986). In addition to this, NK cells may also express receptors which recognize viral peptides or bind to antibodies on the infected cell surface’s (Mace 2023). This makes the NK cell a key player in antiviral immunity by having the potential to eliminate infected cells earlier in infection than antigen dependent cells like cytotoxic T lymphocytes.

NK cells are well known to limit disease progression in viral infections such as HIV-1. However, NK cells are largely heterogeneous, with effector maturation depending on the presence of cytokines, interferon, and other molecules in areas such as secondary lymphoid tissues (Yu et al. 2013). The tonsils are thought to be of particular importance to the maturation and development of early subsets of NK cells (Shannon & Mace, 2021). However, the role of tonsil-resident NK cells in the context of an early viral infection such as HIV-1, which uses lymphoid tissue as a reservoir, is not fully described. Here, Perea and colleagues investigate the role tonsil-resident NK cells play in limiting HIV-1 during early infection.

Key Findings

Characterisation of tonsillar resident NK repertoire

To explore the functional profile of the resident NK cell repertoire, the authors first profiled the resident innate lymphocytes (ILCs) and NK cells in normal human tonsillar tissue, constituting a small proportion of total lymphocytes within the tissue. They identified 11 different subsets classified based on surface expression of CD56, CD16, and tissue residency markers, including CD103, CD69, CD49a, and memory NK cell markers NKG2C and KIRs, with CD16- CD69 single-positive cells constituting the largest subset. Across the different subsets, the authors identified co-expression of CD49a on CD16- NK cells to correlate with elevated baseline degranulation capacity measured by CD107 expression and the ability to produce IFN γ upon co-culture with K562 lymphoblastic cells on CD16+ NK cells. Further, they recognised another functionally responsive subset with a polyfunctional profile defined by KIR  co-expression on CD16- cells.

Since previous studies have shown that HIV can induce heightened expression of HLA-E on lymphocytes  (Natterman, 2005), the ligand for inhibitory NKG2A receptor as a strategy to dampen NK cell cytotoxicity, the authors compared the expression of different inhibitory receptors. Comparative studies of surface expression of inhibitory receptors NKG2A, LAIR-1, PD1, TIGIT, and KLRG1 revealed variability in expression across the different subsets, potentially acting as an immunoregulatory mechanism to prevent overactivation.

Extracellular matrix interactions influence the functionality of resident NK cells

Resident immune populations are retained within peripheral tissues due to interactions between integrin receptors like CD49a and their affinity for extracellular matrix components like collagen. These interactions are especially crucial within the draining secondary lymphoid tissues as they prevent lymphocyte egress, ensuring activation and proliferation of naïve immune cells to combat infection. To expand on this premise, the authors demonstrated increased degranulation capacity via upregulated CD107 expression and IFN γ production on the Collagen I and Collagen IV matrix in the major NK subsets.

Resident tonsillar NK cell responsiveness to HIV infected CD4+ T cells

Moving on, the authors investigated the responsiveness of these functional NK cell subsets towards HIV infected CD4+ T cells by employing an ex vivo tissue explant infection model. Subsets expressing inhibitory receptors, specifically NKG2C, were shown to correlate with a lower HIV burden. An intriguing observation was that while most inhibitory receptors were associated with reduced infection control, LAIR-1 expression exhibited an inverse effect, suggesting it may be a promising area for further investigation. Further, the cytotoxic capacity of sorted CD69+ NK cells was found to be enhanced in a collagen-based 3D co-culture assay, reinforcing the impact of the tissue microenvironment and matrix interactions in NK cell functionality.

HIV infection impedes immune signalling pathways while upregulating inhibitory receptors in tonsil-resident ILC/NK cells

Finally, the study progressed to evaluate global transcriptional changes upon ex-vivo HIV_Bal infection in the identified subsets using single cell RNA sequencing. 11 clusters were identified that genotypically aligned with some of the previously defined subsets, including functionally active CD16- and CD16+ triple positive  NKG2C+ cells. These cells were shown to express cytokine receptors IL2RB, IL4R, IL10RA etc., along with cytotoxic effectors granzymeB, granulysin, and perforin, aligning with their previous characterization as the functionally active subset. Pseudotime inference analysis revealed diverse maturation states likely originating from the activated ielC1 lineage and transitioning towards both immature and differentiated states. Upon DEG analysis of HIV infected explants, the majority of the subsets showed downregulation of immune signaling pathways, including FOS, JUN, and NFKB, impaired IL-15 production, along with downregulation of genes involved in cell adhesion and migration, directing towards an immunologically dysfunctional state. This was further strengthened by a lack of expansion of the broadly defined clusters upon infection, barring the innate lymphocytes ieLC1s, which underwent slight expansion. They further observed a significant reduction in degranulation capacity and increased immune checkpoint expression in the functionally active population upon K562 stimulation and HIV infection.

Figure 6E:  Transcriptome analysis reveals HIV infection impedes immune signalling pathways, including the NFKB, FOS, AND JUN, while upregulating inhibitory receptors indicative of a dysfunctional phenotype in different polyfunctional NK cell clusters. Image made available under a CC-BY 4.0 International license.

Conclusion

Overall, the findings described in this preprint indicate that HIV targets the functionally-active, resident NK cells to dampen the immune response, while the immature states were shown to display immune-stimulatory signatures. This study lays the foundation for exploring the potential of these resident NK populations in HIV infection to harness their cytotoxic effects and to supplement antiretroviral therapy.

What we like about this preprint

This preprint does a thorough job of highlighting the diversity of NK cell functionality based on differential expression of tissue residency, memory, and activating/inhibitory receptors. The reported findings lay the foundation for the further exploration of lymphoid tissue resident NK cells in articulating therapeutic strategies for HIV treatment in early stages of infection. Further, this study accentuates the impact of matrix interactions on NK cell activity and presents a promising model for further investigation.

Questions for the authors

1. Are you planning to investigate differences in resident NK cells to see how they respond to HIV in pediatric vs. adult lymphoid tissues.
2. How would you investigate the mechanisms by which the ECM components influence NK cell function specific to HIV infection?
3. How would you validate the transcriptional findings across larger cohorts and in the different lymphoid tissues?
4. How would you expand on the findings to further investigate the potential of targeting specific immune checkpoints on resident NK cells to enhance antiviral activity during early HIV infection?
5. Are you planning any potential future studies focused on the CD56^– subset of NK cells within the tonsils?
6. We are curious to know how the pseudotime inference analysis performed and what was the basis of selecting ieLC1s as the precursor population for trajectory tracking?
7. Have you considered testing the cytotoxic activity of different NK cell subsets through other functional readouts apart from surface marker expression?

References

• Björkström, N.K., Strunz, B. & Ljunggren, HG. Natural killer cells in antiviral immunity. Nat Rev Immunol 22, 112–123 (2022). https://doi.org/10.1038/s41577-021-00558
• Dogra, P., Rancan, C., Ma, W., Toth, M., Senda, T., Carpenter, D. J., Kubota, M., Matsumoto, R., Thapa, P., Szabo, P. A., Li Poon, M. M., Li, J., Arakawa-Hoyt, J., Shen, Y., Fong, L., Lanier, L. L., & Farber, D. L. (2020). Tissue Determinants of Human NK Cell Development, Function, and Residence. Cell, 180(4), 749–763.e13. https://doi.org/10.1016/j.cell.2020.01.022
• Lanier, L. L., Cwirla, S., Federspiel, N., & Phillips, J. H. (1986). Human natural killer cells isolated from peripheral blood do not rearrange T cell antigen receptor beta chain genes. The Journal of experimental medicine, 163(1), 209–214. https://doi.org/10.1084/jem.163.1.209
• Shannon, M. J., & Mace, E. M. (2021). Natural Killer Cell Integrins and Their Functions in Tissue Residency. Frontiers in immunology, 12, 647358. https://doi.org/10.3389/fimmu.2021.647358
• Yu, J., Freud, A. G., & Caligiuri, M. A. (2013). Location and cellular stages of natural killer cell development. Trends in immunology, 34(12), 573–582. https://doi.org/10.1016/j.it.2013.07.005
• Nattermann J, Nischalke HD, Hofmeister V, Kupfer B, Ahlenstiel G, Feldmann G, Rockstroh J, Weiss EH, Sauerbruch T, Spengler U. HIV-1 infection leads to increased HLA-E expression resulting in impaired function of natural killer cells. Antivir Ther. 2005;10(1):95-107. doi: 10.1177/135965350501000107. PMID: 15751767.
• Freud, A. G., Becknell, B., Roychowdhury, S., Mao, H. C., Ferketich, A. K., Nuovo, G. J., Hughes, T. L., Marburger, T. B., Sung, J., Baiocchi, R. A., Guimond, M., & Caligiuri, M. A. (2005). A human CD34(+) subset resides in lymph nodes and differentiates into CD56bright natural killer cells. Immunity, 22(3), 295–304. https://doi.org/10.1016/j.immuni.2005.01.013

Tags: hiv, immunity, nk cells, therapeutics

doi: https://doi.org/10.1242/prelights.40470

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