CD4 T cell contact drives macrophage cell cycle progression and susceptibility to lentiviral transduction

Macrophages are typically quiescent cells residing in G0, though tissue macrophages have been shown to proliferate locally in tissues; we previously demonstrated that differentiated monocyte derived macrophages (MDM) can be stimulated to re-enter G1 phase of the cell cycle from G0, without cell division. Entry into G1 correlates with an increase in CDK1 expression which phosphorylates the deoxynucleotide-triphosphate hydrolase SAMHD1 at position 592. SAMHD1 not only regulates cellular dNTP levels, but is also a restriction factor for virus replication of HIV-1 and DNA viruses. Here we show that contact with autologous CD4 T cells leads to antigen-independent macrophage cell cycle progression from G0-G1, accompanied by expression of cell cycle associated proteins, including CDK1, and the activation of the canonical MEK-ERK pathway. Further, macrophage cell cycle progression can be blocked not only by anti-cancer drugs targeting the MEK-ERK axis such as Palcociclib, but also by pre-treatment with EGFR antibody, providing additional evidence for cell surface interactions driving proliferative responses. Cell contact with uninfected CD4 T cells renders macrophages ten-fold more susceptible to transduction with VSV-G pseudotyped HIV-1 particles.


Introduction
Macrophages are cells implicated in a diverse array of functions such as defence against pathogens, tissue repair /homeostasis, and anti-cancer activity 1,2 .In addition, macrophages play a role as antigen presenting cells for the adaptive immune response.As such macrophages form 'immunological synapses' with CD4 T cells during MHC II dependent antigen presentation.These involve contact between the two cells via a number of interacting surface proteins including LFA-1 and ICAM-1 in addition to peptide loaded MHC interaction with the cognate TCR on CD4 T cells.Cell polarisation and reorganisation/protein clustering occurs after continual and repeated contact 3 in order to facilitate signalling in the T cell 4 and triggering of an immune response that includes release of IL2 5 .Much less is known regarding antigen independent contact between macrophages and T cells, although they can reciprocally respond to signals from each other to induce inflammatory responses.
Both T cells and macrophages are targets for HIV-1 6 ; cell cycle progression renders both cell types susceptible to infection via CDK-mediated phosphorylation of SAMHD1, a cellular deoxynucleotide-triphosphate hydrolase and lentiviral restriction factor 7,8 .Whilst T cells often proliferate in response to stimulation and become highly susceptible to HIV-1, macrophages typically reside in G0 and are rather refractory to HIV-1 6,9 .
We previously demonstrated a laboratory-based method to stimulate cell cycle progression from G0 to G1 phase in monocyte-derived macrophages (MDM) via activation of the canonical Ras-Raf/MEK/ERK mitogen associated pathway, with an increase in CDK1 expression 9 .More recently we showed that low oxygen environment can also drive cell cycle progression via HIF2a activation of the same mitogen associated pathway 10 .By contrast, danger signals such as 11 and cytotoxic agents 12 can limit cell cycle progression in macrophages and block lentiviral transduction 9 .Here we investigated physiological means by which macrophages may be activated to enter the cell cycle and become more susceptible to transduction.Given known transmission of HIV from T cells to macrophages 13,14 , we hypothesised that infection of macrophages may be facilitated by contact with CD4 T cells as found in tissue reservoirs of HIV such as lymph nodes 15 and spleen.

Results
Monocytes were derived from blood apheresis cones of healthy individuals by adhesion in tissue culture plates before treatment with MCSF to induce differentiation into macrophages for 6 days.Cells were further exposed for 2 days to control conditions (M0), stimulated with LPS/IFNgamma to induce M1 macrophages or IL4/IL-13 to induce M2 polarised macrophages.A fourth group of cells were co-cultured with autologous CD4+ T cells.
Monocyte-derived macrophages were then washed to remove CD4 T cells and either (i) stained for cell surface (polarisation) markers (ii) infected with HIV-1 VSV-G pseudotyped virus (PV), or (iii) stained for markers of cell cycle progression.Cell surface macrophage polarisation markers were investigated under each condition (Figure 1A).As expected M1 polarised macrophages upregulated CD80 and CD86 (Figure 1B); M2 macrophages expressed higher levels of CD86 and CD209 compared to unstimulated macrophages.Whilst macrophages co-cultured with autologous CD4 T cells did not show clear polarisation into either M1 or M2 macrophages, there was upregulation of CD209 similar to that observed in M2 macrophages.
Macrophages co-cultured with CD4 T cells were an order of magnitude more susceptible to infection compared to M0 macrophages (Figure 1C), co-incident with increased expression of intracellular MCM2 (Figure 1D) in co-cultured macrophages.Western blot analysis confirmed higher cellular expression of MCM2 under co-culture conditions and also demonstrated increased CDK1 expression and SAMHD1 phosphorylation with little change in overall SAMDH1 expression (Figure 1E), changes that are associated with cell cycle progression in MDM 9 .
We next performed more extensive analysis of cell cycle status to quantify cell cycle progression markers Geminin, MCM2 and Ki67 (Figure 1F,G).In this analysis, THP-1 monocytic tumour cells demonstrated high expression of all 3 markers, consistent with actively dividing cells.Co-cultured macrophages demonstrated significantly higher expression of MCM2 and Ki67 compared to M0 macrophages, indicating G0 to G1 progression.The modest elevation in Geminin expression was not statistically significant, supporting minimal progression to S, and G2/M.We next sought to establish whether direct cell-cell contact was required for CD4 T cells to induce cell cycle progression in macrophages.Using a transwell system (Figure 1I) we observed that placement of a barrier between macrophages and T cells abrogated the increased susceptibility to infection with PV particles, as well as the cell cycle progression (Figure 1J-K).Immunoblot confirmed that the transwell prevented an increase in MCM2, CDK1 and pSAMHD1 protein levels following the addition of CD4 T cells (Figure 1L).We conclude that direct cell contact is required for T cells to induce cell cycle progression and relieve viral restriction in macrophages.
In order to elucidate pathways that might be involved in mediating CD4 T cell contact dependent cell cycle progression in macrophages we performed phosphoproteomic analysis.
Three time points were assessed: 0, 5 and 120 minutes following contact between cells and cell cycle progression in macrophages was separately confirmed by immunoblot at 2 days timepoint (Figure 2A-B).A number of phosphoproteins were either upregulated or downregulated as demonstrated by volcano plots with the largest number of proteins changing between 0 and 120 minutes (Figure 2C).Gene Ontology and Ingenuity pathway analysis showed that the frequently altered phosphoproteins were involved in signal transduction pathways (Figure 2D).Given this result, activation of signal transduction and our previous work recognising the importance of the MEK/ERK pathway in the activation of macrophage G0-G1 transitioning [REF], we hypothesise that MEK/ERK pathway is involved in CD4 T cell contact dependent cell cycle progression in macrophages.To test whether this pathway was responsible for the cell contact phenotype we used inhibitors of MEK (U0126), CDK4/6 (Palcociclib) and CDK1 (RO-3306).Each of the inhibitors almost completely inhibited MCM2, CDK1 and pSAMHD1 expression following cell-cell contact, indicative of cell cycle arrest in G0 (Figure 2E).This was reflected in concomitant blockade of infection with HIV-1 PV following drug treatment (Figure 2F).
Growth factors and mitogens use the canonical Ras-Raf/MEK/ERK signalling cascade to transmit signals from their receptors to regulate gene expression [REF].EGFR is one of the most important receptors in this pathway [REF].We sought to test whether this receptor could be involved in initiating macrophage cell cycle progression.We therefore treated macrophages with a monoclonal antibody to EGFR prior to co-culture with CD4 T cells.The antibody prevented cell cycle progression even in the presence of CD4 T cells based on the expression of cell cycle associated markers MCM2 and CDK1, and pSAMHD1 (Figure 2E,G).This suggests that EGFR signalling may contribute to cell cycle state in macrophages, and that it can be activated to induce potent cell cycle progression following CD4 T cell contact.

Discussion
These experiments demonstrating cell cycle progression following antigen independent T cell contact delineate a new phenotype of macrophage, with distinct distribution of cell surface marker distribution from M0/M1/M2.Phosphoproteomic analysis revealed that within hours of contact, cell cycle associated proteins in the Ras-Raf-MEK-ERK axis were activated, and this was confirmed biochemically through inhibition of this pathway using immunoblot.
EGFR appeared to mediate the phenotype, consistent with cell-cell signalling.The cell cycle cascade in macrophages resulted in G0-G1 transition as demonstrated by nuclear staining for Ki67 and MCM2, and deactivation of the antiviral activity of SAMHD1.Tissues such as lymph nodes are characterised by close cell contact between T cells and macrophages 15 .The deactivation of SAMHD1 antiviral activity following cell contact may underlie observations that lymph nodes remain a difficult to treat HIV reservoir even in the face of antiretroviral therapy 16 .

Figure 1 :
Figure 1: Direct contact with CD4+ T cells drives macrophage cell cycle re-entry with deactivation of SAMHD1 antiretroviral activity A. Experimental design.Differentiated macrophages were co-cultured with activated autologous CD4+ T-cells, or stimulated by LPS/Interferon gamma (to polarize cells into M1 macrophages), or IL-4/IL-13 (into M2 macrophages).CD4+ T-cells were washed off 2 days later and cell cycle, viral permissivity was analysed.A representative example of microscopic field.* macrophages; scale bar:20um.B. Cell surface markers of macrophage polarization.Macrophages were stained for 6 different markers of polarization and analysed by flow cytometry.M0 (un-treated macrophages) are non-stimulated M-CSF differentiated cells; M0_CD4 (M0 macrophages after co-culture with T-cells); M1 (LPS/Interferon gamma ) and M2 (IL-4/IL-13) macrophages.C. Macrophages were infected with VSV-G pseudotyped HIV-1 expressing GFP.Cells were fixed 2 days post-infection and percentage of GFP positive cells was quantified by using automated microscopic platform.Graphs represent average of n=15 (M0, M0_CD4); n=6 (M1, M2) biological replicates.Statistical analysis was performed using one-way ANOVA with Dunnett's multiple comparisons test.ns, non-significant; * p < 0.1; * * p < 0.01.Bars indicate mean with SD.

Figure 2 :F.I.
Figure 2: Direct cell-to-cell contact drives cell cycle re-entry in human macrophages through EGF receptor and canonical MEK/ERK signalling.