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. Author manuscript; available in PMC: 2013 Dec 5.
Published in final edited form as: Virology. 2012 Oct 3;434(1):68–77. doi: 10.1016/j.virol.2012.09.009

Tubular Cell HIV-Entry through Apoptosed CD4 T cells: A Novel Pathway

Priyanka Singh 1, Hersh Goel 1, Mohammad Husain 1, Xiqian Lan 1, Joanna Mikulak 1, Ashwani Malthotra 1, Saul Teichberg 1, Helena Schmidtmayerova 1, Pravin C Singhal 1
PMCID: PMC3667410  NIHMSID: NIHMS409167  PMID: 23040891

Abstract

We hypothesized that HIV-1 may enter tubular cells by phagocytosis of apoptotic fragments of HIV-1-infected T cells infiltrating tubular interstitium. The study was designed to evaluate the interaction of programmed death-1(PD-1) receptors on CD4 T cells and programmed death ligand-1(PD-L1) on tubular cells (HK2 and HRPTEC, primary tubular cells). Co-cultivation of HIV-1 infected lymphocytes (HIV-LY) with HK2s/HRPTECs resulted in T cell apoptosis, uptake of the apoptosed HIV-LY by HK2s/HRPTECs, tubular cell activation and HIV expression. Cytochalasin-B inhibited tubular cell HIV-LY uptake and anti-PD-L1 antibody inhibited HIV-LY apoptosis and tubular cell HIV-LY uptake, activation and HIV expression. These observations do indicate induction of apoptosis of T cells due to interaction of PD-1 and PD-L1 upon co-cultivation and subsequent phygocytosis of HIV-laden apoptotic bodies by tubular cells and thus the transfer of HIV-1 into tubular cells. These findings identify a novel pathway that facilitates HIV-1 entry into tubular cell.

Renal biopsy data suggest that HIV-associated nephropathy (HIVAN) is the outcome of HIV-1 infection of kidney cells (Bruggeman and Nelson, 2009; Marras et al., 2002; Mikulak et al, 2010). However, kidney cells do not carry conventional HIV-1 receptors (Eitner et al., 2000); therefore, the route of HIV-1 entry into renal cell remained was not clear for a long time (Marras et al., 2002). Recently, enodocytic pathway for HIV-1 entry has been demonstrated to be an important mode of infection in several cells (Dezzutti et al., 2001; Li et al., 2007; Meng et al., 2003; Vidricaire et al., 2003). We previously reported that HIV-1 could enter in both tubular cells and podocytes through endocytic pathways (Hatsukari et al, 2007; Mikulak et al., 2009 and 2010; Mikulak and Singhal, 2010). Moreover, kidney cells were able to transmit HIV-1 particles to co-cultivated T cells and macrophages (Hatsukari et al., 2007). However, endocytosed viral particles in kidney cells persisted for a limited period only. These findings suggested that kidney cells did not provide an ideal milieu for viral replication in in vitro studies. Since renal cell infection has been demonstrated to be an important feature of HIVAN, we hypothesized that activated tubular cells may be either developing or borrowing a suitable milieu for the replication of HIV-1 in vivo. We further hypothesized that the activated tubular cells were capable of phagocytosing T cells and/or fragments of the apoptotic T cells (containing endosomes with viral particles). The latter might have provided a suitable milieu for HIV-1 replication in the host cells.

Epithelial cells have been demonstrated to phagocytose apoptotic lymphocytes both in vivo and in vitro studies (Walsh et al, 1999; Golpon et al., 2004; Willermain et al., 2002). However, renal tubular epithelial cells have not been credited for occurrence of this phenomenon to this date. It did not seem to be lack of studies by the investigators (Patel et al., 2010). For example, recently, potential of tubular cell phagocytic capabilities was studied through their interaction with the apoptotic tubular cells (Patel et al., 2010). Mouse tubular cells were co-cultivated with apoptotic mouse tubular cells and their interaction was compared with macrophages co-cultivated under similar conditions. Macrophages were able to phagocytose the apoptotic tubular cells but mouse tubular cells did not. In these studies, tubular cells could not have phagocytosed the apoptosed tubular cells because of their comparable size. On the other hand, the apoptotic lymphocytes which were several folds smaller in size than of epithelial cells were easily phagocytosed by epithelial cells (Walsh et al, 1999; Golpon et al., 2004). We proposed that tubular cell phagocytosis of the apoptotic/HIV-infected lymphocytes (A-HIV-LY) served as a novel pathway for HIV-1 entry. We also propose that phagocytosis of A-HIV-LY provided a critical milieu to HIV to replicate in tubular cells.

Programmed cell death (PD)-1 is a receptor that is expressed on activated T and B cells (Agata, et al., 1996; Nishimura and Honjo, 2001; Guidotti et al., 1996). PD-1 works by cross-linking with its ligands: PD-L1 and PD-L2. PD-L1 is not only constitutively expressed by T cells, B cells, macrophages, dendritic cells (DC), and several parenchymal cells, including renal tubular epithelial cells (Agata, et al., 1996; Nishimura and Honjo, 2001; Sette et al., 2001; Shimizu et al., 1998; Isogawa et al., 2005; Freeman, et al., 2000; Dong et al., 1999; Yamazaki et al., 2002; Brown, et al., 2003) but is also further up-regulated after activation (Dong et al., 2003). On the other hand, PD-L2 expression is limited to the activated monocytes/macrophages and DCs (Dong et al., 2003). Several studies suggest of an important role for PD-L1 in the regulation of T cell tolerance (Eppihimer et al., 2002; Rodig et al., 2003; Liang et al., 2003); however, the role of PD-L2 is not clear (Agata et al., 1996; Rodig et al., 2003; Liang et al., 2003; Koga et al., 2004). In earlier studies, both PD-L1 and PD-L2 were reported to have either inhibitory or stimulatory effects on T cell responses (Freeman, et al., 2000; Dong et al., 1999). However, recent reports have been consistent with an inhibitory role for PD-L1 (Freeman, et al., 2000; Brown, et al., 2003; Koga et al., 2004; Blank et al., 2004; Mazanet and Hughes, 2002; Barber, 2006; Dong et al., 2004; Latchman et al., 2004). PD-L1 expression in kidneys has been suggested to serve as a regulatory mechanism for limiting activities of autoreactive lymphocytes. Ding et al., (2005) demonstrated that PD-L1 expression by renal tubular cells was associated with suppression of T cell cytokine synthesis.

In the past, several investigators demonstrated transmission of HIV-1 from T cells to other cells including tubular cells or vice versa through synapse formation (Jolly et al., 2010; Muratori et al., 2007; Chen et al., 2011). Recently, Chen et al., (2011) also demonstrated that HIV-infected lymphocytes were able to transmit HIV to tubular cells through virological synapse formation. In our experimental design, we have excluded transmission of HIV through virological synapse formation during co-cultivation studies by using either A-HIV-LY or fragmented A-HIV-LY. We have demonstrated that tubular cell phagocytic pathway may be a novel pathway for the uptake of viral particles contained in the apoptotic/HIV-1 infected T cells or its fragments. Since we have previously reported studies pertaining to the involved mechanism of direct HIV-1 entry into tubular cells through endocytic pathway and rescue of the endocytosed viral particles by immune cells (Hatsukari et al., 2007; Mikulak et al., 2009), we have not investigated that aspect in the present study. We also evaluated the involved mechanism for the induction of apoptosis in HIV-1-infected T cells during their interaction with tubular cells. In addition, we have studied the effect of HIV-1 uptake on the activation of tubular cell downstream signaling as well as phagocytosis associated ROS generation (respiratory burst). Lastly, we were able to establish causal relationships between the induction of HIV-LY apoptosis and their uptake by tubular cells as well as between the HIV-LY phagocytosis and tubular cell activation.

Results

CD4 T cells express PD-1 and renal tubular cells express PD-L1

We hypothesized that tubular cell and T cell interaction induces T cell apoptosis through the activation of PD-1/PD-L1 pathway. CD4 and CD8 T cells were negatively isolated from fresh PBMC obtained from various donors. Analysis of PD-1 in CD4 and CD8 T cells demonstrated 11.6 and 2.7% PD-1 expression respectively and treating them with HIV-1 for two h did not increase PD-1 significantly (Fig. 1a). To determine the expression of PD-L1, HK2 cells and HRPTEC were labeled with isotype antibody and anti-PD-L1 antibody, and evaluated for PD-L1 expression by flow cytometry. Both HK2 and HRPTEC showed expression of PD-L1 approximately 21.8 and 26.07% respectively (Fig. 1b).

Figure 1. Detection of (a) PD-1 expression on CD4 and CD8 and (b) PD-L1 expression on HK2 and HRPTEC cells.

Figure 1

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a. CD4 and CD8 T cells were negatively isolated from PBMC and evaluated for expression of PD-1 following activation. Normal or HIV-treated CD4 and CD8 cells were stained with anti-PD1 labeled with FITC followed by analysis on FACS Calibur. Approximately, 12% of CD4 cells showed PD-1 expression whereas CD8 cells had only about 2%. HIV-1 had no significant effect on PD-1 expression. Results are means ± SD from three sets of experiments.

b. HK2 or primary human proximal tubular (HRPTEC) cells stained with anti-PD-L1 labeled with PerCP or corresponding mouse isotype (IgG1-PerCP) were analyzed on FACS Calibur. Approximately 22% HK2 and 26% HRPTECs showed expression of PD-L1. Results are means ± SD from three sets of experiments.

Co-cultivation of PBMC with renal tubular cells causes depletion of CD4+ T cells

Co-cultivation of normal (uninfected) and HIV-infected PBMC with renal tubular cells for 3 days and subsequent analysis of PBMC for CD4 population by FACS revealed decrease in CD4 cells upon co-culture. Separate co-cultivation experiments were carried out with HK2 and HRPTEC renal cells. In both the experiments, significant decrease in CD4 population was observed upon co-culture with normal PBMC as well as HIV-infected PBMC. Normal PBMC and HIV-infected PBMC incubated alone or co-cultured with HK2 cells were harvested after 3 days and analyzed by FACS for %CD4 population. CD4 population in normal PBMC was 44.8% and after co-cultivation with HK2 cells, it reduced to 31.3% (p<0.001) while in HIV-infected PBMC, CD4 population was 30.3% and after co-cultivation with HK2, it was further reduced to 22.6% (p<0.001) (Fig. 2a). Thus, there was a significant decrease in CD4 population (25–30%) upon co-cultivation of normal or HIV-infected PBMC with HK2 cells. To confirm further, the same experiments were repeated with primary human renal tubular cells (HRPTEC). It was observed that CD4 population in normal PBMC was 47.6.8% and after co-cultivation with HRPTEC, it reduced to 40.7% (p<0.01), while in HIV-infected PBMC, CD4 population was 20.7% and after co-cultivation, it further reduced to around 12.03 % (p<0.001) (Fig. 2b). The decrease in CD4 population upon co-cultivation of normal or HIV-infected PBMC with HRPTEC was around 15 and 41% respectively. Interestingly, with HRPTEC the HIV-infected PBMC showed two and half times higher depletion of CD4 cells, which may be due to their more primary characteristics that may mimic better to in vivo situation.

Figure 2. Co-cultivation of activated normal or HIV-infected PBMC with (A) HK2 and (B) HRPTEC cells and evaluation of CD4 depletion after 5 day of incubation.

Figure 2

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a. Normal or HIV-infected PBMCs were incubated alone or co-cultured with HK2 cells. At the end of incubation period, PBMCs were harvested and stained with anti-CD4 labeled with APC and analyzed by FACS Calibur for CD4 positive cells. HIV-infected PBMCs co-cultivated with HK2 cells showed lowest CD4 positive cells (22%) (highest depletion) while significant decrease in CD4 count was also observed in HIV-CD4(30%) as well as normal CD4+HK2 cells(31%) compared to normal CD4 cells alone(44%). Results are means ± SD from three sets of experiments. *P<0.001 compared to respective CD4

b. Control or HIV-infected PBMCs were incubated alone or co-cultured with HRPTECs. Subsequently, PBMCs were harvested and stained with anti-CD4 labeled with APC and analyzed for CD4 positive cells. Similar phenomenon was observed with HRPTEC cells, HIV-CD4+HRPTEC(12%), HIV-CD4(21%), CD4+HRPTEC(31%) compared to normal CD4 alone(47%). Results are means ± SD from three sets of experiments. *P<0.01 compared to CD4 alone;**P<0.001 compared to HIV-CD4

PBMC and renal tubular cell interaction promotes apoptosis in CD4+ T cells

To prove that depletion of CD4s during co-cultivation of PBMC with tubular cells happens through the induction of apoptosis in CD4 cells, the same experiments as described above were repeated. PBMC were co-stained with anti-CD4 APC labeled antibody and PE Annexin V, and 7-AAD and subsequently analyzed by FACS for double stained cells. As shown in Fig. 3a, after co-cultivation with HK2 both normal CD4 and HIV-infected CD4 T cells showed increased Annexin staining (27.3%) compared to normal CD4 incubated alone (17.1%, p<0.01) and 54.3% compared to HIV-CD4 alone 36.04% (p<0.001). Almost similar results were obtained in co-cultivation experiments with HRPTEC. after co-cultivation with HRPTEC both normal CD4 and HIV-infected CD4 T cells showed increased Annexin staining compared to normal CD4 incubated alone (co-cultivated, 26.3% vs. alone, 15.9%, p<0.01) HIV-infected CD4 alone (co-cultivated, 36.3% vs. alone, 22.1%, p<0.001) (Fig. 3b). These results do indicate that interaction of T cells with renal tubular cells during co-cultivation promotes apoptosis in CD4 T cells, which we speculate to be communicated through interplay between PD-1 and PD-L1. The data also show the increased apoptosis in CD4 cells when HIV-infected PBMC are co-cultivated, which may be due to the fact that HIV itself is a factor for apoptosis and co-cultivation may further intensify the apoptotic signal.

Figure 3. Detection of Apoptotic CD4 T cells by Annexin V after co-cultivation with (a) HK2 and (b) HRPTEC for 5 days.

Figure 3

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a. Normal or HIV-infected CD4 cells incubated alone or co-cultured with HK2 cells. PBMCs were harvested and stained with anti-CD4 labeled with APC, Annexin V-PE and and 7-AAD-PerCP followed by analysis of CD4 subpopulation for Annexin positivity by FACS Calbur. HIV-CD4 co-cultivated with HK2 cells showed highest annexin positive cells (54%) while significant annexin positive CD4 were also observed in HIV-CD4(36%) as well as normal CD4+HK2 cells(27%) compared to normal CD4 cells alone(17%). Results are means ± SD from three sets of experiments. *P<0.01 compared to CD4 alone; **P<0.001 compared to HIV-CD4

b. Similar trend was observed in co-cultivation with HRPTEC cells, HIV-CD4+HRPTEC (36%), HIV-CD4 (22%), CD4+HRPTEC(26%) compared to normal CD4 alone(15%). Results are means ± SD from three sets of experiments. *P<0.01 compared to CD4 alone; **P<0.001 compared to HIV-CD4

c. PD-L1 blocking by anti-PD-L1 Ab significantly decreased annexin staining after co-cultivation of HIV-CD4 with tubular cells (apoptotic death of CD4 due to interaction of PD1 and PD-L1). Results are means ± SD from three sets of experiments. *P<0.01 vs. CD4 alone; **P<0.001 vs HIV-CD4 alone; ***P<0.01 vs. HIV-CD4+HK2+Iso.

CD4 T cell apoptosis in co-cultivation is induced by PD-1 and PD-L1 interaction

To confirm the role of PD-1/PD-L1 pathway in the induction of T cell apoptosis, HK2 and HRPTEC were pretreated with anti PD-L1 antibody (15 μg/ml) or isotype IgG1 (15 μg/ml) for one hour before co-cultivation. CD4+ T cells were negatively isolated from PBMC and subsequently infected with HIV-1 and co-cultured with HK2 and HRPTEC. Co-culture was maintained for 3 days, T cells harvested, stained for apoptosis with PE Annexin V, and 7-AAD and then analyzed by flow cytometry. PD-L1 blocking in HK2 cells reduced Annexin positive CD4 cells from approximately 34.3% (untreated or isotype) to 26.5% (p<.0.01) while in HRPTEC, Annexin positivity in CD4 cells was decreased from around 27% (untreated or isotype) to 21.6% (p<.0.05, data not shown) (Fig 3c). It appears that PD-L1 blocking reduced CD4 T cell apoptosis significantly suggesting a role of PD-1 and PD-L1 interaction in CD4 population when they come in contact with renal tubular cells.

Phagocytic uptake of apoptotic T cells or fragments thereof by renal tubular cells

The above studies provided the experimental proof that once CD4 cells come in contact with renal tubular cells, some of them undergo apoptosis and one of the major factors that trigger apoptosis is the interaction of PD-1 and PD-L1. The published literature suggests that cells with phagocytic properties are activated when they come in contact with apoptotic cells or their fragments and thus they phagocytose apoptotic cells or their fragments. Therefore, we hypothesized that apoptotic T cells or fragmented bodies get phagocytosed by renal tubular cells facilitating transfer of HIV-1 or parts of it (proteins and genomic material). To evaluate the role of phagocytosis of apoptotic T cells by renal tubular cells, Jurkat T cells (Clone E6-1) were transduced with VSV envelop pseudotyped NL4-3: ΔG/P-GFP (NL-GFP/LY) virus for 72 hours followed by trypsinization and repeated washing to remove adherent viral particles. Subsequently, these cells were apoptosed and fragmented as mentioned in material and methods and then co-cultivated with HK2s for 24 h. Non-transduced cells were used as a control. Co-cultivated HK2s were washed and then examined under confocal and fluorescent microscopes. Representative micrographs displaying GFP expression under confocal microscope are shown in Fig. 4a. In parallel sets of experiments HK2 were co-cultivated with either with GFP tagged empty vector (EV) - or NL4-3 pseudovirus for 72 hours. Subsequently cells were examined under a fluorescent microscope. A total of 820 cells were counted; 33.6 ± 3.2% cells showed presence of GFP (Fig. 4b). These findings indicate that HK2s are able to acquire apoptotic GFP+ bodies/HIV proteins or genomic particles upon co-cultivation.

Figure 4. Phagocytic uptake of apoptotic T cells/fragments by tubular cell.

Figure 4

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a. HK2 cells co-cultivated with either buffer (control) or NL-GFP/LY (apoptosed and fragmented) for 72 h and then examined under confocal microscopy after extensive repeated washing. Representative microphotographs of control HK2 and HK2 co-cultured with with NL-GFP/LY are shown. Tubular cell uptake of GFP positive T cells fragments indicates transfer of HIV proteins/genome.

b. HK2 cells co-cultivated with either empty vector (EV)GFP/LY or NL-GFP/LY for 72 hours. Subsequently, cells were repeatedly washed and examined under a fluorescence microscope. GFP +ve cells were counted.

c. The same HK2 cells analyzed by FACS for GFP. Uptake of GFP indicated phagocytic uptake and transfer of HIV proteins/genome into tubular cells.

d. JTLRG-R5 cells infected with full length NL4-3 virus or primary HIV-1 HT/92/599 or infected were apoptosed and fragmented and then co-cultured with HRPTEC for 24 h. After removing T cells and extensive washing of adherent HRPTEC, they were observed under confocal microscope. HRPTEC co-cultured with HIV-infected T cells displayed green punctuate bodies indicating the uptake of lymphocyte fragments containing viral particles. Representative microphotographs (phase, left panel), fluorescent (middle panel) and combined (right panel) are shown.

HK2s treated under similar conditions were also assayed for GFP+ cells by flow cytometry. Representative flow scans of control HK2s and NL4-3/GFP/LY are shown in Fig. 4c.

Phagocytic uptake and expression of HIV-specific RNA expression in HRPTEC

To determine the phagocytic uptake of apoptotic T cells by primary renal tubular cells (HRPTEC), JTLRG-R5 (X4 and R5 receptor expressing cells) were infected with laboratory grown full length NL4-3 virus or primary HIV-1HT/92/599. These T cells upon HIV-infection express GFP and thus can be monitored visually under fluorescent microscope. After 3–5 days post-infection, these cells as well as control cells without infection were induced for apoptosis and then fragmented followed by co-culture with HRPTEC for 24 h. The HRPTEC then washed extensively with PBS-ETDA (4 times) and observed under confocal microscope. The HRPTEC co-cultured with HIV-infected JTLRG-R5 cells showed parts of T cells that were green under fluorescent light while control cells did not show any green fluorescent Fig. 4d.

Apoptosis plays a central role in phagocytic uptake of T cells/fragments by tubular cells

To confirm the role of T cell apoptosis in general and PD-1/PDL-1 mediated apoptosis in particular, HK2 cells were pre-treated with either buffer (control), anti-PD-L1 antibody (15 μg/ml), or caspase-3 inhibitor (5 μM) for 30–60 min, followed by co-cultivation with NL-GFP/LY for 24 hours. At the end of the incubation, cells were analyzed by flow cytometry for GFP expression. As shown in Fig 5a, anti-PD-L1 antibody inhibited HIV detection by 60%; whereas, caspase-3 inhibitor reduced it by 85%. These findings indicate HK2 cells phagocytosed predominantly apoptotic cells, in which apoptosis was predominantly triggered through PD-1/PD-L1 pathway. However, a small percentage of HIV-1-infected T cells might have also undergone apoptosis through different pathways.

Figure 5. Role of apoptosis and phagocytosis in tubular cell HIV uptake.

Figure 5

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a. To confirm that apoptosis is the key step for T cell uptake by tubular cells, anti-PD-L1 antibody or caspase-3 inhibitor or buffer was added in HK2 cells for 30 min. Thereafter, NL-GFP/LY as such were used for co-cultivation for 24 h. After incubation, T cells were removed and HK2 cells were washed extensively with PBS-EDTA and then analyzed by flow cytometry for GFP expression. GFP positive HK2 population treated with anti-PD-L1 antibody or caspase-3 inhibitor was significantly reduced compared to without treatment. *P<0.01 control vs anti-PD-L1; **P<0.001 control vs. caspase-3 inhibitor.

b. To assess the role of phagocytosis in transmission of HIV into tubular cells, co-cultivation of HK2 with apoptosed and fragmented HIV-infected PBMC was done in the absence or presence of cytochalasin-B (10 μM) for 24 h. Subsequently, total RNA was extracted and expression of Gag was measured (as a marker of HIV replication) by real time PCR. Significant inhibition of HIV-1 expression was observed in the presence of cytochalain-B. *P<0.01 compared with control.

Role of phagoctyosis in tubular cell HIV uptake

To confirm the role of phagocytosis in the establishment of productive HIV infection in tubular cells, apoptotic HIV-infected lymphocytes (JTLRG-R5 infected with HIV-1HT/92/599) were fragmented and then incubated with HK2 in the media containing either buffer (control) or cytochalasin B (cyto-B, 10 μM) for 24 hours. Cells were washed with PBS-EDTA. Subsequently, total RNA was extracted from HK2 and probed for gag transcripts by real time PCR. As shown in Fig. 5b, Cytochalasin B inhibited (P<0.01) Gag expression in HK2 cells. These findings indicate that phagocytosis of lymphocyte fragments contributed to HIV transmission expression in tubular cell.

HIV-specific RNA expression in HK2

To determine the transmission and replication of HIV in HK2 cells, control T cells or NL-GFP/LY were apoptosed by heating method followed by fragmentation. The fragmented T cells were co-cultivated with HK2 for 24 hours. Thereafter, cells were washed with PBS-EDTA to remove any adherent lymphocyte or its fragments. Total RNA was extracted from HK2s and probed for Nef by RT-PCR. As shown in Fig. 6a, co-cultivated HK2s showed Nef expression.

Figure 6. HIV-specific RNA expression in tubular cells.

Figure 6

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a. Normal and HIV-1 HT/92/599 infected PBMC (apoptosed by heating) co-cultivated with HK2 for 24 hours followed by repeated washing with PBS-EDTA. Subsequently, total RNA was extracted from tubular cells and analyzed for Nef expression by RT-PCR. Lane 1 shows a molecular marker, lane 2 shows HK2 co-cultivated with uninfected LY, lanes 3, 4, and 5 show HK2s co-cultivated with HIV-infected PBMC and lane 6 shows HIV-infected PBMC alone (positive control).

b. Spliced form of HIV-1 transcripts (Tat-402, Rev-219/225, Nef-203) were evaluated from the above total RNA. Lane 1, molecular marker; lanes 2, HK2+HIV-PBMC (test sample), lane 3, HK2 +normal BPMC (control) and lane 4, HIV-infected PBMC alone (positive control). Visible DNA bands were obtained for Rev-219/225 and Nef-203 in lanes 2 and 4, however, Tat-402 was not visible; lanes 5, 6, and 7 are same as lanes 2, 3 and 4 respectively, but PCR from RNA without RT; lanes 8,9 and 10 are same as lanes 2,3, and 4 respectively for GAPDH RT-PCR.

To further confirm HIV replication in tubular cells, the total RNA extracted from above HK2 cells was analyzed for multiply spliced RNA expressed in early human immunodeficiency virus type 1 infection by RT-PCR. As shown in Fig 6b, HK2s displayed mRNA expression of multiply spliced RNA in tubular cells and thus indicating HIV replication

Morphologic evaluation of the phagocytosed T cells and their fragments

To visualize the uptake of the fragments of apoptotic lymphocytes by HK2s, HK2s were co-cultivated with NL-GFP/LY for 24 h, and then repeatedly washed with PBS-EDTA, followed by staining with acridine orange. Cells were examined under a confocal microscope. Individual cells attempting encircle T cells are shown in Fig 7 (a, b, and c). Many of tubular cells contained only part of T cell nuclei (Fig 7e and 7f). Electron microscopic studies confirmed tubular cell uptake of T cells. A representative electron microphotograph is shown in Fig. 7d.

Figure 7. Morphologic evaluation of tubular cell endocytosed fragments of lymphocytes.

Figure 7

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HK2 co-cultivated with NL-GFP/LY for 24 hours, and then repeatedly washed with PBS- EDTA, followed by examination under microscope.

a, b, and c. Representative microphotographs of an individual tubular cell showing uptake of lymphocyte or its fragments (indicated by arrows) observed under fluorescent microscope.

d. Cells treated under above conditions were prepared for electron microscopic studies. A representative electron micrograph showing the presence of lymphocyte nucleus (indicated by an arrow) within a tubular cell.

e and f. Cells prepared under above mentioned conditions were stained with acridine orange and then visualized by confocal microscopy. Lymphocyte nuclear fragments (arrows) are displayed within HK2s.

g and h. HK2 and NL-GFP/LY were labeled with CFDA (green) and CMTMR (orange) dyes respectively. Subsequently co-cultivated co-cultivation was carried out for 4 hours, followed by washing of cells with PBS-EDTA. HK2 were evaluated under fluorescence microscope for uptake of lymphocyte fragments. Representative fluorescence microphotographs showing presence of orange and yellow dots (indicated by arrows) within HK2s.

Next, to confirm intracellular localization of lymphocyte fragments in tubular cells, NL-GFP/LY and HK2 cells were labeled with CMTMR (orange) and HK2s with CFDA dyes, respectively. Subsequently, labeled NL-GFP/LY were co-cultivated with labeled HK2 for 4 h and washed with PBS-EDTA extensively. HK2s were evaluated under a fluorescent microscope for double labeling as a marker of tubular cells uptake of lymphocyte fragments. Presence of yellow dots indicates intracellular localization of lymphocyte fragments in tubular cells (Figs. 7g & 7h).

Exclusion of the role of synapse in the transmission of HIV from T cells to HK2s

Recently, Chen et al., reported T cell transmission of HIV through virological synapse formation (Chen et al., 2011). To exclude the role of virological synapse, the Jurkat T cells transduced with NL4-3:3G/P-GFP virus or JTLRG-R5 cells infected with full length were apoptosed by heating method and then fully NL4-3 or primary HIV-11HT/92/599 fragmented. Fragmented cells were co-cultivated with HK2 or HRPTEC as described above for 24 hours; washed with PBS-EDTA and analyzed by flow cytometry, confocal microscopy or RT-PCR. HIV transmission through viral synapse has been reported in earlier studies, in the present study, however, the possibility of viral synapse formation was excluded by induction of apoptosis and fragmentation of T cells. These results thus suggest the transfer of HIV into tubular cells and its expression via phagocytosis of apoptotic bodies.

Activation of tubular cells during interaction with apoptotic lymphocytes

Macrophages have been reported to display phosphorylation of AKT after phagocytosis of the apoptotic cells (Patel et al., 2010). To determine whether tubular cells also behave like professional phagocytic cells, HK2s were incubated with either HIV-1 infected PBMCs (HIV-LY) or buffer for 24 hours and washed with PBS-EDTA. As shown in Figs. 8a and 8b, lysates by immunoelectrophoresis in HK2s, showed enhanced expression of phospho-AKT when compared to control cells.

Figure 8. Activation of tubular cells during interaction with apoptosed HIV-LY.

Figure 8

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a. HK2s were incubated with either HIV-1 infected PBMCs or buffer for 24 hours (n=3). Subsequently, HK2 were prepared for Western blotting and probed for phsopho(P)-AKT and Actin. The upper panel shows phospho-AKT expression by HK2 under control (C1, C2, and C3) and experimental condition (HIV1, HIV2, and HIV3). The lower panel shows actin content of cells under similar conditions (n=3).

b. A cumulative bar diagram showing phosphor (P) -AKT:Actin ratio of 3 sets of experiments.

c. HK2s were incubated with either infected PBMCs or buffer for 24 hours. Subsequently, cells were washed with PBS-EDTA and then loaded with MitoTracker green and Red CC1 and then evaluated for ROS generation. HK2/HIV-PBMC displayed enhanced ROS generation in the form red fluorescence when compared with control cells. Majority of the cells also displayed orange and yellow fluorescence indicating that both mitochondrial and cytosolic NADPH oxidases participated in ROS generation.

d. HK2 were pretreated with either anti-PD-L1 antibody, or cytochalasin B and then incubated with HIV-PBMC for 24 hours. Subsequently, cells were loaded with MitoTracker green and Red CC1 and then examined under a fluorescence microscope for detection of ROS generation. Anti-PD-L1 antibody displayed minimal ROS generation. Similarly, pretreatment of HK2s with cytochalasin B completely attenuated tubular cell ROS generation.

Since macrophage phagocytic event has been shown to be associated with respiratory burst in the form of ROS generation (Robinson, 2009), we studied tubular cell ROS generation during the uptake of HIV-LY. HK2 cells were incubated with either infected PBMCs or buffer for 24 hours and washed with PBS-EDTA. Cells were loaded with MitoTracker green (to display mitochondrial localization) and Red CC1 (to display ROS generation) and examined under a fluorescence microscope for detection of ROS generation. As shown in Fig. 8c, HK2/HIV-LY displayed enhanced ROS generation in the form red fluorescence when compared with control cells. However, majority of the cells also displayed orange and yellow fluorescence indicating that both mitochondrial and cytosolic NADPH oxidases participated in ROS generation.

To determine a link between occurrence of respiratory burst, apoptosis of HIV-LY and uptake of the apoptosed HIV-LY, HK2 were pretreated with either anti-PD-L1 antibody or cytochalsin B and then incubated with HIV-LY for 24 hours. Subsequently, cells were loaded with MitoTracker green and Red CC1 and then examined under a fluorescence microscope for detection of ROS generation. As shown in Fig. 8d, Anti-PD-L1 antibody partially inhibited tubular cell ROS generation. These findings suggested that inhibition of T cell apoptosis was also associated with the attenuated tubular cell resipiratory burst. Similarly, pretreatment of tubular cells with cytochalasin B completely attenuated HIV-induced respirtatory burst (Fig. 8d).

Discussion

The present study demonstrates a novel pathway for tubular cell HIV-1 entry from the infected T cells. It also highlights the mechanism which facilitates the tubular cells to serve as HIV-1 reservoir. Interaction of kidney with the infected T cells promoted apoptosis of T cells. Since anti-PD-L1 antibody prevented HK2/HRPTEC-induced T cell apoptosis, it suggested that HK2/HRPTEC-induced T cell apoptosis was mediated through PD-1 and PD-L1 interaction. Occurrence of apoptosis not only prompted tubular cell uptake of the apoptotic-infected T cells (A-I/TCs) or their fragments but also facilitated HIV-1 expression by tubular cells. Since both anti-PD-L1 antibody and caspase-3 inhibitor prevented tubular cell HIV expression it appeared that induction of apoptosis in infected T cells was critical for tubular cell HIV expression. Imaging studies confirmed that tubular cells had phagocytosed the apoptotic T cells or their fragments. Moreover, cytochalasin B, an inhibitor of phagocytosis, not only inhibited tubular cell uptake of A-I/TCs but also attenuated HIV-1 expression. In addition, tubular cell uptake of A-I/TCs was associated with upregulation of AKT and enhanced ROS generation (respiratory burst). Furthermore, both anti-PD-L1 antibody and cytochalasin B inhibited tubular cell ROS generation (respiratory burst). These findings indicated that the induction of apoptosis of PD-1+/CD4 T cells prompted tubular cells to phagocytose them and thus facilitated not only the activation of tubular cells but also provided exposed them a critical milieu which was suitable for HIV replication.

Several investigators suggested that PD-1 and PD-L1 inhibit the immune response during viral infections (Nishimura and Honjo, 2001; Freeman et al., 2000). Studies on PD-1 knockout mice infected with adenovirus showed enhanced clearance of adenovirus from the liver; the latter was attributed to increased proliferation of CD4+ and CD8+ T cells in the absence of PD-1 signaling (Dong et al., 2004). In another study, Barber et al., showed that viral titers in mice infected with LCMV were undetectable in the spleen and liver and significantly reduced in the lungs and kidneys following the treatment with the PD-L1 mAb; the latter suggested that PD-1 and PD-L1 interaction led depletion of CD4 cells by apoptosis (Latchman et al., 2004). In the present study, we demonstrated that interaction of PD-1 expressing T cells with PD-L1 expressing HK2s led to the apoptosis of T cells. Since anti PD-L1 antibody attenuated HK2-induced T cell apoptosis, it confirmed the role of PD-1: PD-L1 pathway during this interaction. It appears that active killing of infected T cells by tubular cells in vivo may be serving as an auto-clearance mechanism of immune cells in general and HIV-1-infected cells in particular. However, this phenomenon carries a trade off- granting tubular cells an HIV-1 reservoir status.

The present study indicates that tubular cells are capable of phagocytosing the apoptotic T cells and their fragments. Many of the fragments may be composed of endosomes containing HIV-1. It appeared that the uptake of these endosomal compartments might have provided a critical milieu to HIV-1 for their replication in the host cells. However, further studies are needed to evaluate the exact mechanism involved in providing a suitable milieu or a critical factor (s) by T cell endosomes carrying viral particles in tubular cells.

Macrophages play a major role in the removal of apoptotic and necrotic cells in vivo (Krysko et al., 2006; Maderna and Godson; 2003; Hart et al., 1996). They are so proficient in removal of apoptotic cells that at times it had been difficult to find apoptotic cells in biopsy tissue specimens (Hart et al., 1996). Apoptotic cells develop specific activity which directs wandering cells such as macrophages to come into their vicinity and phagocytose those (Krysko et al., 2006). On the other hand, tubular cells are fixed cells and they can only phagocytose populations of cells wandering in their vicinity such as immune cells or malignant cells (Walsh et al., 1996; Golpon et al., 2004; Willermain et. al., 2002). In a recent in vitro study, tubular cells got activated and initiated down regulation of AKT, after coming in contact with the apoptotic tubular cells (Patel et al., 2010); whereas, phagocytosis of the apoptotic tubular cells by macrophages was associated with an up regulation of AKT (Patel et al., 2010). In the present study too, phagocytosis of the apoptotic T cells by tubular cells was associated with the upregulation AKT. Thus, it appears that surface interaction of tubular cells with the apoptotic cells might be downregulating AKT; whereas, phagocytosis of the apoptotic cells by tubular cells was associated with upregulation of AKT. From that perspective tubular cells behaved similar to professional phagocytes such as macrophages. In addition, tubular cell uptake of A-I/TCs was associated with respiratory burst, which was akin to macrophage phagocytic profile.

Since majority of HK2/HIV-LYs displayed respiratory burst it appears that all cells were activated; on the other hand only 33% of HK2s showed HIV expression. This discrepancy may be related to the uptake of the infected vs. non-infected fragments HIV-LY. Interestingly, pretreatment of tubular cells with anti-PD-L1 antibody not only provided protection to HIV-LY against apoptosis but also prevented tubular cell ROS generation; these findings indicated that PD-1:PD-L1-induced T cell apoptosis was essential for tubular cell ROS generation. Since anti-PD-L1 antibody also prevented tubular cell HIV-1 expression it further confirmed that PD-1: PD-L1- induced apoptosis and subsequent phagocytosis by HK2s was critical for tubular cell HIV-1 expression. In the present study, pretreatment of tubular cells with cytochalasin B not only prevented tubular cell respiratory burst but also provided protection against HIV expression. These findings suggested that phagocytosis of the apoptosed HIV-LY was a pre-requisite both for ROS generation and tubular cell HIV expression.

In summary, we have highlighted the role of phagocytic properties of tubular cells which allow them to serve as an HIV-1 reservoir. The latter status of tubular cells can be blocked by either blocking PD-1:PD-L1 pathway by anti-PD-L1 antibody or by inhibiting tubular cell phagocytosis of the apoptotic lymphocytes (Fig. 9). We conclude that tubular cell triggered HIV-infected T cell apoptosis plays a critical role in tubular cell uptake of T cells and tubular cell activation.

Figure 9. Schematic diagram showing proposed mechanism allowing tubular cells to serve as an HIV-1 reservoir.

Figure 9

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T cells express PD1 and HK2 cells express PD-L1. In phase 1, HIV-1-infected T cells come across with adjacent tubular cells. This interaction allows cross-linking of PD-1 and PDL-1, which promotes T cell apoptosis. In phase 2, the activated tubular cells phagocytose the apoptotic/infected T cells or their fragments. Since viral particles are maintained in T cell milieu, they are able to fuse with T cell enodosomal membranes (in the host cells) and thus they are able replicate in tubular cells. HIV-1 reservoir status of tubular cells can be blocked by either blocking PD-1:PD-L1 pathway by anti-PD-L1 antibody or by inhibiting tubular cell phagocytosis of the apoptosed lymphocytes by cyotochalasin-B.

Experimental Procedures

Cells and Viruses

Peripheral blood mononuclear cells from healthy donors undergoing leukopheresis were separated on a Ficoll-Hypaque gradient. To eliminate monocytes, cells were incubated for 2 hrs in PRIMARIA flasks (Falcon, BD Biosciences). PBMCs (nonadherent cells) were collected and resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and stimulated with 5μg/ml of phytohemagglutinin for three days. CD4 or CD8 cells were negatively isolated from PBMCs as per manufacturer’s instructions (InvitrogenDynal, Norway). Lymphocytes were infected with primary X4 strain HIV-1HT/92/599 or laboratory adapted HIV-1 stain NL4-3 for 2 hours, followed by extensive washing. Afterwards, cells were cultivated in medium containing 20U/ml of IL-2 (Roche, Indianapolis, IN). HK2s were obtained from ATCC and human renal proximal tubular epithelial cells (HRPTEC) from ScienceCell (Carlsbad, CA). HRPTECs were used up to 15 passages as recommended by the provider. In co-cultivation studies, HK2s/HRPTECs were plated and lymphocytes infected with HIV-1 or uninfected were added next day in a ratio of 1:2 followed by incubation at 37 C for three to five days.

Production of Viral Stocks

Primary X4 Viral stocks (HIV-1HT/92/599, AIDS reagent program, NIH) were prepared in PHA-activated primary lymphocytes cultivated in the presence of IL-2 (48). Viral stocks were also prepared for laboratory adapted HIV-1 NL4-3 by transfection of full length clone of pNL4-3 in 293T cells. The supernatant was harvested after 48 h, filtered through 0.45 μm pore size syringe filters, aliquoted and stored at −80°C. The virus titers were determined using p24 ELISA kit (Clontech, Mountain View, CA). Replication defective and VSV envelope pseudotyped HIV-1 stocks expressing GFP were produced from pNL4-3: G/P-GFP and empty vector (pHR’-CMV-CMV-IRES-GFP B) constructs as described previously (45).

Analysis of surface and apoptotic markers by Flow Cytometry

Expression of PD1 on CD4 and CD8 T cells and PD-L1 on renal proximal tubular cells (HK2 and HRPTECs) were measured by staining them using mouse anti-human PD1 labeled with FITC (eBioscience) and mouse anti-human PD-L1 labeled with PerCP (eBioscience) respectively or respective isotype control antibodies as per manufacturer’s instructions followed by analysis on FACS Calibur (Becton Dickinson) and Cell Quest software.

To measure the depletion of CD4 T cells, normal or HIV-infected PBMC were co-cultivated with HK2 and HRPTEC or incubated alone for 3 days. Thereafter, the PBMC were removed, washed with PBS and stained with APC mouse anti-human CD4 (BD Pharmingen) and analyzed by FACS as described above.

To confirm that the depletion of CD4 cells in co-cultivation experiments was happening through the interaction of PD1 on CD4 cells and PD-L1 on renal tubular cells, the same experiments described above were repeated and PBMC were stained with annexin V-PE and 7-AAD along with APC mouse anti-human CD4 and then analyzed within one h for percentage of CD4 cell apoptosis by flow cytometry as mentioned above. For blocking Study, HK2s or HRPTEC were incubated with anti-PD-L1 antibody or isotype IgG1 (e-bioscience) at a concentration of 15 μg/ml for one hour before co-cultivation with HIV-infected PBMC. Cells were maintained in co-culture for 3 days and then stained and analyzed by flow cytometry as done earlier.

Determination of the role of apoptosis in uptake of T cell fragments by tubular cells

To evaluate the role of phagocytosis of apoptotic cells or its fragments by tubular cells, normal or NL-GFP/LY (NL4-3:3G/P-GFP-transduced lymphocytes) were facilitated to undergo apoptosis by heating at 45°C for two hours, followed by incubation at 37°C for four hours. Subsequently, these cells were co-cultured with HK2s for 72 hours (n=3). At the end of the incubation period, cells were washed with PBS containing 0.4 mM EDTA four times (to detach the adherent lymphocytes and lymphocyte fragments). Cells were prepared for confocal microscopy as well as for FACS analysis to observe number of GFP+ve cells (as a marker phagocytic uptake by tubular cells).

Morphologic evaluation of phagocytic uptake of T cell fragments by renal tubular cells

To determine the uptake of the fragmented/apoptosed uninfected (LY) or infected (NL-GFP/LY) by renal tubular cells (HK2), tubular cells were co-cultured with either LY or NL-GFP/LY for three days. At the end of the incubation period, cells were repeatedly washed with a buffer containing EDTA. Subsequently, cells were stained with acridine orange (49) and then examined for the uptake of T cell fragments under a confocal microscope. Further, to confirm intracellular localization of lymphocyte fragments in tubular cells, double labeling was performed. LY or HIV-LY were labeled with orange (5,6-[4-chloromethyl]-[benzoyl-amino]-tetramethylrhodamine, CMTMR, cat no. C2927, Molecular Probes) and tubular cells with green (5,6-carboxyfluorescein diacetate succinimidyl ester, CFDA, cat no. C1157, Molecular Probes) dyes. In brief, cells were incubated in media containing respective dye (5 μM) for 30 min. Subsequently, cells were washed and kept in dye-free media for 12 hours. At the end of the period, control and color labeled were co-cultured for 4–6 h, followed by repeated washing with PBS-EDTA. Thereafter, tubular cells were examined under fluorescence microscopy for double labeling as a marker of tubular cells uptake of lymphocyte fragments.

Exclusion of the role of synapse in the transmission of HIV from T cells to tubular cells

HIV-infected GFP reporter cells were apoptosed by heating method and then fragmented by passing repeatedly through 25 gauge needle. Cell lysate was centrifuged at high speed. Complete fragmentation of the cells (cell debris) was confirmed by examining an aliquot under a light microscope. LY (control) and cell debris were co-cultivated with HK2/HRPTEC for 24 hours. At the end of the incubation, tubular cells were repeatedly washed with buffer containing EDTA and processed for FACS analysis and PCR studies for HIV-1 expression as mentioned above.

Phagocytic uptake of T cell/fragment by tubular cell by Electron microscopy

HK2s were incubated with HIV-LY for 24 hours. Subsequently, cells were fixed in 10% glutaraldehyde and prepared for electron microscopic studies as reported earlier (9).

Phagocytic uptake of T cell fragments by tubular cells facilitates HIV-transmission

PBMC/JTLRG-R5 GFP reporter cells were infected with NL4-3 laboratory adapted virus or primary HIV-1 virus (HIV-1HT/92/599) as mentioned elsewhere. These cells turn green upon HIV-1 infection. To determine the uptake of HIV by tubular cells during co-cultivation, control (LY) or HIV-infected lymphocytes (HIV-LY) were apoptosed and fragmented as described above and then co-cultivated with HK2 or HRPTEC for 3 days, followed by removal of lymphocytes by repeated washing with PBS containing 0.4 mM EDTA. Subsequently, tubular cells were observed under confocal microscope for uptake of T cells fragments or total RNA was isolated and tubular cell expression of Nef as well as spliced form of early transcripts (Tat-402, Rev-219/225, Nef-203) were determined by RT-PCR as evidence of HIV-1 uptake and replication.

Determination of the role of endocytosis in uptake of HIV-LY fragments by tubular cells

Cytochalasin B is known to inhibit endocytosis by disrupting actin filament (Axline and Reaven EP, 1974). To determine the role of endocytosis, HIV-LYs were co-cultivated with HK2s in the media with or without cytochalasin-B (10 μM) for 24 hours. At the end of the incubation period, cells were washed with PBS-EDTA extensively to remove HIV-LYs followed by total RNA extraction from both the sets of HK2 cells. RNA was assayed for the presence of Gag transcripts by real time PCR in both sets.

RT-PCR and Quantitative PCR Analysis

Total RNA was extracted from control and experimental HK2s using TRIZOL (Invitrogen corp.). For cDNA synthesis, 2 μg of the total RNA was preincubated with 2 nmol of random hexamer (Invitrogen Corp) at 65°C for 5 min. Subsequently, 8ul of the reverse-transcription (RT) reaction mixture containing Cloned AMV RT, 0.5 mmol each of the mixed nucleotides, 0.01 mol dithiothreitol, and 1000 U/mL Rnasin (Invitrogen Corp.) was incubated at 42°C for 50 min. For a negative control, a reaction mixture without reverse transcription (RT) was used. Samples were subsequently incubated at 85°C for 5 min to inactivate the RT. Primer sequences for HIV-1 Nef were 5′-ATGGGTGGCAAGTCAAAACG-3′ (forward) and 5′-TCAGCAGTCTTTGTAGTACTCCG-3′ (reverse). To amplify multiply spliced form of HIV-1 RNA transcripts (Tat-402, Rev-219/225, Nef-203), the following primers were used: us, 5′-TCTCTCGACGCAGGACTCGGCTTGC-3′ (forward) and Art-7, 5′-TTCTATTCCTTCGGGCCTGTCG-3′ (reverse) (46). The amplification conditions were as follows: Denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and polymerization at 68 °C for 2 min. For internal control, GAPDH was used as mentioned earlier (47).

Quantitative PCR for Gag was carried out in an ABI Prism 7900HT sequence detection system using following primer sequences: 5′-GGAGCTAGAACGATTCGCAGTTA-3′, (forward) and 5′-GGTGTAGCTGTCCCAGTATTTGTC-3′, (reverse). SYBR green was used as the detector and ROX as the passive reference dye (47). Results (means ± S.D.) represent three experiments as described in the legend. The data was analyzed using the Comparative CT method (ΔCT method). Differences in CT are used to quantify relative amount of PCR target contained within each well. The data was expressed as relative mRNA expression in reference to control, normalized to quantity of RNA input by performing measurements on an endogenous reference gene, GAPDH (47).

Activation and generation of ROS in tubular cells during co-cultivation

HK2 cells were cultured alone or co-cultured with HIV-infected (NL4-3) PBMC for 24 h followed by extensive washing by PBS-EDTA and then lysed in RIPA buffer. Western blot analysis was done from these protein lysates to detect phosphor-Akt (Cell Signaling Technology, Beverly, MA) and actin as described previously (48). To evaluate ROS generation, PBMC were removed from co-culture and extensively washed with PBS-EDTA. The HK2 cells then were loaded with Mito Tracker green (100nM, to label mitochondria) and Red CC1(0.5 μM, to display ROS generation) (Molecular Probe, InVitrogen) for 15 min and after washing with PBS, the cells were analyzed under confocal microscope using FITC filter for green fluorescence and rhodamine filter for red fluorescence. To block PD1 and PD-L1 interaction or endocytosis, the HK2 cells were pretreated with PD-L1 antibody (eBioscience) for 1–2 h or cytochalasin B for 30 min followed by co-culture with HIV-LY for 24 h and analysis for ROS generation as described above.

Statistical analysis

For comparison of mean values between two groups, the unpaired t test was used. To compare values between multiple groups, analysis of variance (ANOVA) was applied and a Bonferroni multiple range test was used to calculate a p-value. Statistical significance was defined as P<0.05.

Acknowledgments

This work was supported by grants RO1DK084910 and RO1 DK083931 (PCS) from National Institutes of Health, Bethesda, MD. This work was presented at the 42nd Annual Meeting of the American Society of Nephrology, Denver, CO. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: Jurkat E6-1 from Dr. Arthur Weiss, JTLRG-R5 from Olaf Kutsch, and HIV-192HT599 from Dr. Neal Halsey.

Footnotes

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