HDAC inhibitors Inactive in Primary CD4 T Cells? PDF Print E-mail
Written by Alain Lafeuillade   
Thursday, 01 September 2011 00:00

The Use of Histone Deacetylase Inhibitors (HDACi) for Purging HIV Reservoirs Could Be Compromized by Their Lack of Activity in Primary CD4 T Cells Compared to Cell Line Models of HIV Latency

HDACiThis is the conclusion of a study recently published in the Virology Journal (1). Although HDACi have previously demonstrated their ability to reactivate latent HIV in transformed, actively dividing cell lines models of HIV latency, this was not confirmed in latently infected primary CD4 T cells generated in the H80 co-culture system.

We asked Professor Carine Van Lint (University of Bruxelles) to comment these findings.

 

 

T
he results reported in the paper of Sahu GK et al are quite embarassing. Several experiments performed in the last few years have demonstrated the role of chromatin modeling in the maintenance of HIV latency and gave hope for new strategies able to "purge" the HIV reservoir (2). 

 

In this paper (1) the authors tested the activity of valproic acid (VPA), trichostatin A (TSA) and prostratin (which is not an HDACi) to reactivate latent HIV in primary T cells generated in the H80 co-culture system (3). Although prostratin induced a 4 fold increase in HIV production, no effect was observed with VPA or TSA.

 

As the low percentage of latently infecetd T cells in the H80 co-culture system could be a confounding factor in assessing the responsiveness of latent HIV to HDACi, the authors modified their experiment in order to increase the number of latently infected cells in culture. But the same results for prostratin -and the lack of activity for VPA and TSA- were again observed.

The authors point out that the requirements for reactivating latent HIV appear to be multifactorial. In particular, the amounts of NF-kB, CycT1, Cdk9 are essential. 

 

The authors conclude that studying HIV latency in non-dividing normal CD4 T cells should be prefered to the use of transformed, actively dividing cell lines. 

 

W
e asked to Professor Carine van Lint, a well renowned expert in the field, a few questions about the implications of these results. She kindly agreed to explain for us the subtleties of this area of research.

 

Alain Lafeuillade: You have extensively studied the activity of HDAC inhibitors in vitro on cellular models of HIV latency. What are, in you experience, the best candidates to reactivate HIV?
Carine van Lint:
Among HDACIs tested in vitro in HIV-1 latently-infected cell lines, SAHA (Suberoylanilide hydroxamic acid; vorinostat), a member of the hydroxamic acid class of HDAC inhibitors, represents the best candidate today to start clinical trials since it has been approved by the FDA for the treatment of cutaneous T cell lymphoma (4-6).

AL: In the recent paper of Sahu et al (1) no activity was found for valproic acid or trichostatin A in primary T cells. How could you explain their findings?
CVL:  Sahu and Cloyd have recently reported that latent HIV in primary lymphocytes is unresponsive to the histone deacetylase inhibitors VPA and TSA (1). Their model uses purified CD4 T cells from normal donors’ blood stimulated with crosslinked anti-CD3 for 2 days and cultured in IL-2 media for 5 days. Cells are then infected with a CXCR4-dependent low cytopathic HIV strain. The infected cells are cultured for 2-3 weeks and then co-cultured on the H80 feeder cell line for 6 weeks. Infected cells were tested for their reactivation potential using the HDACIs TSA and VPA in the presence of AZT. The authors did observe an increase of p24 production after prostratin (a PKC agonist) treatment (which is however rather slight) and no effect using VPA and TSA (4). Other groups have developed such a primary cell line model to study HIV-1 latency and tested HDACIs for their reactivation potential in these models (7), as described above.


In the model developed by Sharon Lewin group, resting CD4+ T cells can efficiently be infected after incubation with either CCR7 ligands or CCL19 or CCL21 (8) (an incubation step that did not induce appreciable activation or proliferation). An important contribution of this ex vivo model is the concept that a viral latent state can be achieved through direct infection of a quiescent, resting memory CD4 T cell (indeed, other models use activated cells as targets for infection, which are then allowed to return to a resting state). This group has shown that HDACIs (including SAHA, MCT3, LBH589 and MS275) reactivate HIV from latency in two independent donors but to a lesser extent than the potent activator PMA (data presented at the IAS conference, Vienna 2010).


The group of Siliciano developed a novel model of latency that involves lentiviral transduction of primary CD4+ T cells with a Bcl-2 cDNA to increase cell survival in vitro (9). These cells are then activated with anti-CD3/CD28 antibodies and IL-2, and infected. Latently-infected cells are then obtained after allowing infected cells to return to a quiescent state via incubation in the absence of cytokines. It is unclear whether ectopic expression of Bcl-2 in this system may introduce artifactual effects in terms of cellular activation, differentiation that, in turn, may affect viral latency (9). Using this model, they demonstrated that HIV-1 latency was barely reactivable by VPA (5nM) but the HDACIs TSA allowed viral reactivation at a dose of 200nM.


In the system developed by Karn’s group (10), primary CD4 cells are stimulated with αCD3 + αCD28 antibodies in the presence of IL-2 and then infected with VSV-G-pseudotyped HIV-1 vectors (lacking the env gene and containing either wild-type or attenuated Tat). After 2 days, productively infected cells are purified by flow cytometry sorting and placed into culture in the presence of αCD3 + αCD28 plus rIL-2 for 4–6 weeks. Cells are then co-cultured on H80 feeder cell monolayers in the presence of rIL-2. After approximately 6 weeks, 70–90% of the infected cells harbor latent virus that no longer express the reporter gene (10). Analysis by chromatin immunoprecipitation assays (ChIP) has demonstrated that TCR activation results in increased histone acetylation and recruitment of NF-κB to the LTR promoter region of integrated HIV-1. Exposure to SAHA alone does slightly reactivate viral transcription, but this may be due to limiting levels of P-TEFb in primary T cells.  Moreover, when used in combination with other HIV inducers, SAHA reactivated viral expression in a synergistic manner (11).


Planelles laboratory developed a primary cell models based on the isolation of naïve CD4+ T cells, which are then activated and induced to differentiate into a TCM-like phenotype known as non-polarized cells (NP)(12). Infection with HIV-1 is performed while the cells are in an activated state, and viral latency is strongly favored by the natural progression of activated cells to a quiescent, memory-like state (12). 
In addition, other groups including ours have tested the HDACIs reactivation potential in resting CD4+ T cells isolated from HIV-infected patients under HAART. Resting cells were isolated by negative selection (MACS) from PBMCS of HAART-treated HIV+ patients and cultured in the presence or not of different compounds for 6 days. Viral reactivation was then assessed by measuring HIV RNA in cell supernatants (Amplicor test, Roche). Our laboratory has demonstrated that HDACIs could reactivate HIV from latency and acted synergistically on viral reactivation when used in combination with other HIV inducers (13,14). Moreover, the group of D. Margolis demonstrated that class I HDAC inhibitors were strikingly efficient inducers of virus outgrowth from resting CD4 T cells of aviremic patients, whereas HIV was rarely recovered from patient's cells exposed to class II HDAC inhibitors (15). Of note, the weak HDACI VPA did reactivate viral expression in cells isolated from HAART-treated individuals, but to a lesser extent than the more potent HDACI SAHA (14). 


In conclusion, the lack of viral reactivation observed in primary cell models by certain groups, including Sahu and Cloyd, may rely on the experimental settings. Despite the small divergences between their two primary cell systems, Sahu and Cloyd did not observe viral reactivation after exposure to VPA and TSA, while Karn’s group did show a small but significant viral reactivation upon SAHA treatment (and a higher reactivation when using combinations of SAHA and other HIV inducers) (11). Of note, in vitro primary cell models of HIV latency are more physiologically relevant than chronically-infected cell lines and represent therefore an important tool in the field. However, they need to be improved (see below) and the data obtained with resting T cells isolated from HAART-treated aviremic individuals represent an even more physiological model.

AL: Do you think that our in vitro models of HIV latency are flawed and that it is too early to translate the discoveries they have provided into clinical trials?
CVL: No, I think that, eventhough none of the in vitro primary cell models developed so far is perfect, they do allow to study HIV latency in a more physiological context than transformed chronically infected cell lines. The use of Bcl2 and/or of activators like IL-2, such as the coculture step, represents bias and may impact on the establishment of HIV-1 latency. Further work has to be performed in order to improve and establish more relevant primary cell line model systems and animal models. This further work is a prerequisite to translate reactivation data to clinical trials. However, at least two clinical trials have started with SAHA: one headed by Sharon Lewin, Monash University, Melbourne, Australia and another by David Margolis, University of North Carolina in Chapel Hill, USA. These two trials will undoubtfully bring important and very informative data for future purging clinical trials.


 

References

(1) Sahu GK, Cloyd MW. Latent HIV in primary T Lymphocytes is unresponsive to histone deacetylase inhibitors. Virol J 2011; 8 (1): 400 [epub ahead of print]

(2) Colin L, van Lint C. Molecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategies. Retrovirology 2009; 6: 111

(3) Sahu GK Lee K, Ji J, Braciale V, Baron S, Cloyd MW. A novel in vitro system to generate and study HIV latently-infected long-lived normal CD4+ T-lymphocytes. Virology 2006; 355(2): 127-37

(4) Duvic, M., and Vu, J. (2007) Expert Opin Investig Drugs 16(7), 1111-1120

(5) Marks, P. A. (2007) Oncogene 26(9), 1351-1356

(6) Mann, B. S., Johnson, J. R., Cohen, M. H., Justice, R., and Pazdur, R. (2007) Oncologist 12(10), 1247-1252

Sahu, G. K., and Cloyd, M. W. (2011) Virol J 8(1), 400
(7) Bosque, A., and Planelles, V. (2011) Methods 53(1), 54-61
(8) Saleh, S., Solomon, A., Wightman, F., Xhilaga, M., Cameron, P. U., and Lewin, S. R. (2007) Blood 110(13), 4161-4164
(9) Yang, H. C., Xing, S., Shan, L., O'Connell, K., Dinoso, J., Shen, A., Zhou, Y., Shrum, C. K., Han, Y., Liu, J. O., Zhang, H., Margolick, J. B., and Siliciano, R. F. (2009) J Clin Invest 119(11), 3473-3486

(10) Tyagi, M., Pearson, R. J., and Karn, J. (2010) J Virol 84(13), 6425-6437
(11) Friedman, J., Cho, W. K., Chu, C. K., Keedy, K. S., Archin, N. M., Margolis, D. M., and Karn, J. (2011) J Virol 85(17):9078-89
(12) Bosque, A., and Planelles, V. (2009) Blood 113(1), 58-65
(13) Quivy, V., Adam, E., Collette, Y., Demonte, D., Chariot, A., Vanhulle, C., Berkhout, B., Castellano, R., de Launoit, Y., Burny, A., Piette, J., Bours, V., and Van Lint, C. (2002) J Virol 76(21), 11091-11103
(14) Reuse, S., Calao, M., Kabeya, K., Guiguen, A., Gatot, J. S., Quivy, V., Vanhulle, C., Lamine, A., Vaira, D., Demonte, D., Martinelli, V., Veithen, E., Cherrier, T., Avettand, V., Poutrel, S., Piette, J., de Launoit, Y., Moutschen, M., Burny, A., Rouzioux, C., De Wit, S., Herbein, G., Rohr, O., Collette, Y., Lambotte, O., Clumeck, N., and Van Lint, C. (2009) PLoS One 4(6), e6093
(15) Keedy, K. S., Archin, N. M., Gates, A. T., Espeseth, A., Hazuda, D. J., and Margolis, D. M. (2009) J Virol 83(10), 4749-4756



Key words: HDAC inhibitors, HIV cure, HIV eradication study, HIV latency, HIV reservoir purge, carine van lint, towards an HIV cure
Last Updated on Thursday, 01 September 2011 20:50
 

Smileys

:confused: :cool: :cry: :laugh: :lol: :normal: :blush: :rolleyes: :sad: :shocked: :sick: :sleeping: :smile: :surprised: :tongue: :unsure: :whistle: :wink: 

  1000 Characters left

Antispam Refresh image Case sensitive