Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• HIV latency

• CD4+ memory T cell clones

• Genetic transcription

Main Findings

Antiretroviral treatment improves the longevity of people living with human immunodeficiency virus by controlling HIV-1 replication. However, a latent CD4+ T cell reservoir that harbours HIV provirus escapes ART and thus treatment does not offer a cure (1). This reservoir is responsible for rapid rebound viraemia and inhibits progress in HIV cure (1-3). Challenges in targeting these cells include their rarity and difficulty in characterising their transactional program (4, 5) . The objectives of the preprint by Weymar GH and colleagues was to identify and describe the transcriptional program of CD4+ T cells harboring intact latent provirus and to explore a novel approach using their T cell receptors (TCR) to enrich clones of these latent cells.

The researchers first determined how best the intact latent provirus of interest could be enriched. Cells were purified for CD45-RA expression, gene expression at the TCR-b locus (TRBC) using flow cytometry and functional variable domains identified by step-wise limited dilution cell sorting with env sequencing. After combined enrichment, the latent provirus of interest was found in the range of 6 to 163 in 10^4 CD4+ T cells (median of 16 in 10^4 CD4+ T cells). It is known that HIV-1 provirus can integrate into CD4+ T cells at any point along their differentiation. By comparing the TCR frequencies to the relative frequency of the specific env from proviral DNA in similarly enriched samples, they determined that most clonally expanded CD4+ T cells harbor latent HIV-1.

Next, they sought to identify the precise TCR alpha-beta sequence of the latent clones of interest by using the limiting dilution sorting strategy with sample enrichment based on CD45-RA, TBRC and functional domain expression. The genes of interest identified included TRAV12-3/J9/TRBV5-4/J1-1, TRAV26- 2/J32/TRBV2/J1-1, TRAV38-1/J33/TRBV4-3/J2-3. They then wanted to determine whether CD4+ T cells harboring intact latent provirus share a transcriptional profile. Combining their TCR sequencing data with the 10x genomics mRNA data and using uniform manifold approximation and projection, a statistical technique, they produced 15 unique clusters among CD4+ T cells harboring intact provirus. On average 57 % of all cells expressing the TCR associated with the expanded latent clone were found in a specific cluster (‘cluster 7’). This data then projected on a reference data set of multimodal single cell analysis of peripheral mononuclear blood cells from HIV negative individuals further characterized these latent clone cells of interest to be in the CD4+ T effector memory compartment. This compartment is known to express chemokine receptors that enable cells migration to sites of inflammation (6). Specific cluster-defining, differentially expressed genes included HLA-DR and HLA-DP, CD74, CCL5, Granzymes A and K, cystatin F (CST7), LYAR and DUSP2. The researchers were thus able to meet their initial aim to describe the transcriptional program of the latent clones of interest through their novel methodology. They concluded that expanded clones of latent cells carrying intact HIV-1 provirus persist preferentially in distinct CD4+ T cell populations which is important for progress towards developing a cure for this disease.

Limitations

• This study would have benefitted from functional studies on identified clones

• By only focussing on large expanded clones of latent cells, it is possible that non-circulating CD4+ T cell subsets were excluded and may have a different gene expression

• Within circulating CD4+ T cell subset, it is likely that other populations that still have the clone of interest and were not described in this research.

Significance/Novelty

This research extends previous observations by identifying the transcriptional program of resting latent cells and linking it to specific genes of interest. This is important for the development of curative treatment targeting strategies for HIV. The step-wise methodology was thorough and provided a novel characterisation for these cells of interest. Host-virus dynamics were also considered given that HIV infects CD4+ T cells at different stages of maturation. This research contributes to understanding of HIV latency in aid of progress towards cure.

References

1. Chun, T. W. et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci U S A 94, 13193-13197, doi:10.1073/pnas.94.24.13193 (1997)

2. Finzi, D. et al. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 5, 512-517, doi:10.1038/8394 (1999)

3. Chun, T. W. et al. Rebound of plasma viremia following cessation of antiretroviral therapy despite profoundly low levels of HIV reservoir: implications for eradication. AIDS 24, 2803-2808, doi:10.1097/QAD.0b013e328340a239 (2010).

4. Bachmann, N. et al. Determinants of HIV-1 reservoir size and long-term dynamics during suppressive ART. Nat Commun 10, 3193, doi:10.1038/s41467-019-10884-9 (2019).

5. Crooks, A. M. et al. Precise Quantitation of the Latent HIV-1 Reservoir: Implications for Eradication Strategies. J Infect Dis 212, 1361-1365, doi:10.1093/infdis/jiv218 (2015)

6. Farber, D. L., Yudanin, N. A. & Restifo, N. P. Human memory T cells: generation, compartmentalization and homeostasis. Nat Rev Immunol 14, 24-35, doi:10.1038/nri3567 (2014).

Credit

Reviewed by Lisha Jeena as part of the cross-institutional journal club of the Immunology Institute of the Icahn School of Medicine, Mount Sinai, the Kennedy Institute of Rheumatology and the Oxford Centre for Immuno-Oncology  (OXCIO) (University of Oxford, GB) and Karolinska Institute’s Center for Infectious Medicine (CIM) & Center for Molecular Medicine (CMM).