macrophages are scavengers that phagocytose dead and dying cells during normal tissue homeostasis , and detect and eliminate infected cells in their role as innate immune sentinels ( devitt and marshall , 2011 ; poon et  al . , 2010 ) . in immunodeficiency virus - infected hosts , macrophages may comprise up to 10% of infected cells ( zhang et  al . , 1999 ) , survive for extended periods as a viral reservoir ( gorry et  al . , 2014 ) , and drive infection - related neurological disorders ( burdo et  al . , 2013 ) . tropism of hiv-1 for macrophages is determined both by receptor ( cd4 ) and coreceptor ( ccr5 and cxcr4 ) expression ( r5 and x4 viruses , respectively ) and by additional less well - defined factors ( duncan and sattentau , 2011 ) . viruses transmitted between individuals , termed transmitted / founder ( t / f ) viruses , are minimally tropic for macrophages ( ochsenbauer et  al . , 2012 ; 2009 ) , implying that macrophage infection occurs at a late stage after viral transmission when the virus has adapted to infect macrophages more efficiently . macrophage infection by cell - free hiv-1 is rate limited by fluid - phase uptake ( carter et  al . , 2011 ; marchal et  al . , 2001 ) and low plasma membrane expression levels of viral entry receptors ( lee et  al . , 1999 ) . a mode of retroviral infection of cd4 t  cells that is more efficient than cell - free spread is cell - to - cell spread ( dale et  al . , 2013 ; sattentau , 2008 ) , exemplified by virological synapses ( vss ) and associated structures that drive efficient high - multiplicity infection in  vitro ( dale et  al . , 2013 ; sattentau , 2008 ) and may dominate viral dissemination in  vivo ( murooka et  al . infected macrophages transfer high - multiplicity hiv-1 infection to cd4 t  cells , promoting reduced viral sensitivity to reverse transcriptase inhibitors and some neutralizing antibodies ( duncan et  al . , 2013 ; , 2014 ; gousset et  al . , 2008 ; groot et  al . , however , the principal mechanism by which hiv-1 infects macrophages is unclear , and the ability of hiv-1-infected t  cells to transmit virus to macrophages has not been studied . since cd4 t  cells are proposed to be the major cell type infected by immunodeficiency viruses at transmission and throughout infection ( li et  al . , 2009 ; zhang et  al . , 1999 ) , we investigated interactions between hiv-1-infected t  cells and macrophages to determine whether virus might transfer directly between them . we show that primary monocyte - derived macrophages ( mdms ) selectively capture autologous primary hiv-1-infected cd4 t  cells , leading to infection of mdms that is of greater magnitude than the corresponding cell - free virus infection , particularly for t / f viruses . to investigate whether hiv-1-infected t  cells might interact with macrophages , we cocultured mdm with ccr5-expressing jurkat - tat - ccr5 t  cells ( jurkats ) or primary cd4 t  cells infected with fluorescent x4 ( hiv-1nl4.3-gfp ) or r5 t / f virus ( hiv-1ch077mcherry ) and live - cell imaged over 2  hr . figure  1a shows stills from movie s1 ( available online ) , in which a mdm sequentially engulfs three hiv-1nl4.3/gfp jurkats . similarly , an mdm engulfs two hiv-1ch077/mcherry jurkats ( movie s2 ) or an  hiv-1ch077/mcherry primary autologous cd4 t  cell ( movie s3 ) . these results suggest that mdm capture is selective for hiv-1 t  cells but independent of viral tropism . since mdms appeared to ignore apparently healthy , uninfected t  cells , we hypothesized that mdm might selectively engulf hiv-1 t  cells via direct recognition of cell surface viral antigen and/or indirectly through recognition of t  cell death , since hiv-1 infection induces t  cell death by apoptosis and other mechanisms ( cooper et  al . 2014 ) and macrophages avidly take up dead and dying cells ( devitt and marshall , 2011 ; poon et  al . , we  tested this hypothesis using multispectral flow cytometry ( imagestream ) quantitation of mdm uptake of hiv-1 and/or dead / dying t  cells . an advantage of this technique over conventional flow cytometry is that images can be quantified for capture and internalization of t  cells rather than reporting nonspecific cell  aggregation or engulfment of cell debris by mdms . autologous primary cd4 t  cells were isolated , infected with wild - type ( wt ) r5 hiv-1bal , and processed for imaging as described in  figure  1b . t  cells were labeled prior to coculture with mdms  for markers of apoptosis ( phosphatidylserine [ ps ] ) or late apoptosis / necrosis ( live / dead label [ ld ] ) ( figures 1c and s1a  s1h ) , and within mdm after coculture , washing , lifting , fixation , and permeabilization for cd3 , gag , and active caspase3 ( figures 1d and s1i s1n ) . selective mdm uptake of different t  cell subsets was quantified by expressing the proportion of each subset in the t  cell culture prior to uptake , and within the mdm population subsequent to uptake , as an uptake index ( figures s1i  s1n ) . an index of < 1 indicates t  cells are selectively ignored , whereas an index of > 1 means t  cells are selectively captured from the starting t  cell pool . uninfected ( gag ) healthy ( caspase3/ps / ld ) t  cells were selectively ignored by mdms compared to either infected / healthy ( p  < 0.01 ) or uninfected / dead / dying ( p  < 0.05 ) captured t  cells ( 10-fold increased capture over uninfected/ healthy ) . strikingly , infected / dead / dying t  cells were highly significantly captured by mdms compared to uninfected / healthy ( 50-fold over p  < 0.001 ) and at a significantly higher frequency than either infected / healthy t  cells ( p  < 0.01 ) or uninfected / dead / dying cells ( p  < 0.05 ) ( figure  1e ) . these data demonstrate that cell death and hiv-1 infection independently promote t  cell capture by mdm , but combined they mediate a strong uptake signal . to interrogate short - term interactions mediating hiv-1 t  cell capture by mdm , we quantified t  cell uptake using qpcr of mdm - associated viral ( v)dna ( figure  2a ) or luciferase content using the luciferase reporter hiv-1 infectious molecular clone ( imc ) hiv-1bal - luc ( ochsenbauer et  al . , 2012 ) . hiv-1 t  cells were cocultured with mdm for the times indicated , stringently washed free of unengulfed t  cells , and immediately lysed and assayed . we compared hiv-1 t  cell uptake with cell - free virus using supernatant derived from the same hiv-1 t  cells immediately prior to coculture with mdms or across 3  m transwells that prevent t  cell transfer but allow free diffusion of virus ( figures s2a  s2c ) ( martin et  al . , 2010 ) . vdna copy number was normalized to -globin to account for variability in mdm number , and the signal was further normalized to the 1  hr time point to compensate for interdonor variability . we detected increasing t - cell - associated , but not cell - free , hiv-1 signals in mdms over 6  hr ( figure  2a ) , consistent with cumulative t  cell capture by mdm . to investigate engulfment mechanisms , we blocked actin remodeling , inhibiting cytoskeleton - dependent processes including phagocytosis , or macropinocytosis , using nontoxic or weakly toxic but functional concentrations ( figures s2d  s2f ) of jasplakinolide or amiloride ( eipa , carter et  al . , 2011 ) , respectively . jasplakinolide significantly inhibited ( p  < 0.01 ) hiv-1 t  cell uptake , whereas eipa did not ( figure  2b ) , suggesting phagocytosis , rather than macropinocytosis , as a potential uptake mechanism . to further probe uptake interactions we tested inhibitors of hiv-1 envelope glycoprotein ( env)-receptor and cell death receptor engagement . none of the env - receptor inhibitors ( figures 2b and s3a  s3c ) or published inhibitors of interactions driving phagocytosis of dead / dying cells ( figure  s4 ) reproducibly and significantly blocked hiv-1 t  cell uptake . we further tested the requirement for env - receptor interactions by assaying uptake of cd4 t  cells infected with wt or env - deficient hiv-1 . both populations were equivalently captured ( figures 2c , 2d , s3d , and s3e ) , confirming that hiv-1 t  cell - mdm recognition is env - receptor independent and therefore not mediated via conventional vs signals ( jolly et  al . , 2004 ; sattentau , 2008 ) . potential outcomes of hiv-1 t  cell uptake by mdm include phagolysosomal elimination without mdm infection or viral spread from t  cells to infect mdm . to interrogate this we prepared and cocultured mdm with hiv-1 t  cells ( figure  1b ) , washed and maintained cultures up to 72  hr   azt , and measured vdna content . the signal rose by > 10-fold from baseline without azt demonstrating active hiv-1 replication , whereas with azt it decreased by 3-fold , implying vdna degradation ( figure  3a ) . we compared infection kinetics of mdm directly exposed to hiv-1 t  cells or across transwells and assayed released viral p24 gag representing productive infection . direct coculture of hiv-1 t  cells with mdm for 3  hr or longer yielded robust and prolonged gag release ( figure  3b ) . by contrast , 3  hr exposure of mdm to hiv-1 t  cells across transwells did not result in a spreading infection , and 6 and 12  hr transwell exposure resulted in delayed gag release with substantially lower peak values . although these data imply mdm infection , a proportion of gag  release might result from virus production by mdm - captured hiv-1 t  cells . we therefore investigated whether mdm became infected in the absence of residual t  cells . cd4 t  cells were infected with hiv-1nl4.3-egfp - bal , cocultured with mdm for 3  days to approximate a single - cycle mdm infection , washed , and imaged . we observed 5% mdm with strong cytoplasmic gfp expression in the absence of detectable t  cells ( figures 3c and 3d ) that was eliminated by azt treatment , demonstrating direct mdm infection , whereas cell - free infection yielded an insignificant gfp mdm signal . to further investigate the link between hiv-1 t  cell capture and mdm infection , we maintained cocultures for 3  days following uptake inhibition experiments and measured gag release . jasplakinolide , which reduced hiv-1 t  cell uptake ( figure  2b ) , strongly ( p  < 0.01 ) inhibited t - cell - mediated mdm infection but weakly ( p > 0.05 ) reduced cell - free infection ( figure  3e ) . we tested for inhibition of mdm infection by extended culture of mdms exposed to hiv-1 t  cells , in the continued presence of entry inhibitors . all inhibitors reduced mdm infection ( figure  3f ) , demonstrating that although these reagents do not prevent hiv-1 t  cell capture by mdm ( figure  2b ) , they act at a subsequent step to block viral entry into mdm further confirmation of mdm infection by hiv-1 t  cell capture was obtained using mdms derived from an individual homozygous for the ccr5 32 mutation , which are resistant to r5 hiv-1 infection ( bol et  al . , 2009 ) . these mdms were confirmed ccr5 null compared to cells from wt individuals ( figure  s5a ) . heterologous ccr5 primary cd4 t  cells infected with hiv-1bal - luc were directly cocultured with , or exposed across transwells to , ccr5 or wt donor mdm for 6  hr , washed , cocultured for 3  days , and assayed for luciferase . figure  3 g shows that the ccr5 mdms produced weak signals not significantly above baseline , probably reflecting residual t - cell - associated hiv-1 , whereas the four wt donor mdm signals were 10-fold higher for both direct coculture and cell - free infection , demonstrating mdm infection . to investigate the possibility that hiv-1 transfer from t  cells to mdms yields an abortive infection at a late stage of viral replication , we cocultured mdm with hiv-1 t  cells directly or across transwells for 6  hr , washed , cultured for 3  days , and assayed supernatant infectivity on tzm - bl cells . significantly more infectious virus ( p  < 0.05 ) was released from mdm directly exposed to infected t  cells than exposed to cell - free virus ( figure  3h ) . we also added autologous cd4 t  cells   azt to these mdm to act as indicators of onward cell - to - cell hiv-1 spread from mdms to t  cells . this revealed efficient hiv-1 infection of the reporter t  cells that was greater in magnitude from mdms initially cocultured with infected cd4 t  cells than infected across transwells , and infection was abolished by azt ( figure  3i ) . taken together , these data demonstrate that exposure of mdms to hiv-1-infected cd4 t  cells leads to robust and productive mdm infection that is greater in magnitude , at all time points and under  all conditions tested , than exposure to the cell - free viral counterpart . we hypothesized that the efficiency of mdm infection by hiv-1 cd4 t  cell uptake might influence viral tropism , and we used imagestream to investigate the fate of macrophage ( m)-tropic and nonmacrophage ( nm)-tropic hiv-1-infected t  cells associated with mdm . we confirmed that mdm cocultured with m - tropic hiv-1bal t  cells for 6  days were productively infected : 25% of mdms expressed cytoplasmic gag and released free gag p24 , and these signals were eliminated by azt treatment ( figure  4a and 4b ) . t  cells infected with nm - tropic x4 virus hiv-1iiib were taken up by mdms ( figure  4a ) , as anticipated from the results in figure  1a and movie s1 . however , the majority of the hiv-1iiib gag signal within the mdm was associated with cd3 , suggesting internalized infected t  cells , and the remainder with cd3-negative vesicular compartments possibly representing degraded t  cells that had lost cd3 expression ( figures 4a and 4c ) . by contrast , the gag signal in mdms cocultured with hiv-1bal t  cells was predominantly ( 75% ) not associated with a cd3 signal ( figures 4a and 4c ) , implying infection of 15% mdms in the absence of residual t  cells or debris . however , this analysis only detects cd3 and gag signal within the same mdm and does not differentiate mdms that contain a cd3 signal with a nonassociated cytoplasmic gag signal , which would imply mdm infection in the presence of residual t  cell material . to probe this , we established a brightfield mask to define the mdm and then excluded cd3 regions from this mask using a stringent cd3 mask . this analysis revealed that for hiv-1bal , 22% mdms were gag within which 18% contained non - cd3-associated cytoplasmic gag . for hiv-1iiib 6% mdms were gag within which 4% was non - cd3-associated , a signal not significantly above baseline ( figure  s5b ) . the cd3-associated hiv-1 gag signal within mdms might represent a small population of viable t  cells able to propagate infection , or cd3 debris from degraded hiv-1 t  cells . to interrogate this we created a gate to report only cd3 signal representing an intact t  cell , based on shape and size ( round , > 100 pixels in area , equivalent to 25  m , approximately the area of a t  cell ) , thereby excluding cd3 debris ( figure  s5c ) . we found that 2%3% of all mdms contained intact t  cells , corresponding to 6% of both hiv-1bal and hiv-1iiib - infected mdms . furthermore , when these internalized intact t  cells were analyzed for infection , the majority ( 55%70% ) were gag ( figure  s5d ) . these data reveal that a small proportion of hiv-1-infected mdms contain potentially viable hiv-1 t  cells several days postuptake but that infection of mdm , as reported by a significant proportion of cytoplasmic gag within mdm , only takes place with m - tropic hiv-1 . although hiv-1 transfer from infected t  cells to mdms does not appear to expand the tropism of nm - tropic hiv-1 , it may influence infection efficiency of viruses weakly tropic for mdms . we therefore investigated whether r5 t / f viruses that are predominantly t  cell tropic by cell - free infection ( ochsenbauer et  al . mdms were cocultured directly , or across transwells , with cd4 t  cells infected independently with six different t / f viruses or two chronic m - tropic luciferase - encoding imcs ( figure  s5e ) as before and assayed for infection . cell - free infection by m - tropic hiv-1 imcs gave robust luciferase signals at day 6 ( figure  5a ) and gag release over 10  days ( figure  5b ) . by contrast , cell - free t / f virus mdm infection yielded 10-fold lower levels of luciferase than hiv-1bal and hiv-1yu2 ( figure  5a ) and insignificant gag release compared to baseline over 10  days ( figure  5b ) , consistent with previous reports ( ochsenbauer et  al . 2009 ) . however , direct coculture of t / f virus - infected t  cells with mdms yielded 30-fold greater day 6 luciferase and significantly ( p  < 0.001 ) greater gag release than cell - free  infection ( figure  5b ) . t / f virus infection of mdms was confirmed by imaging mdms at day 6 postcoculture , demonstrating extensive cytoplasmic gag in the absence of intact t  cells , although residual scattered cd3 t  cell debris was evident in some mdms ( figure  5c ) . the enhanced ability of t / f viruses to infect mdms via cell - to - cell spread from infected t  cells might be explained by an increased multiplicity of infection ( moi ) imparted by this mode of spread . to test this we exposed mdm to increasing titers of cell - free m - tropic or t / f imcs and evaluated mdm infection after 3  days . m - tropic hiv-1bal - luc and hiv-1yu2-luc gave robust luciferase signals that increased with input cell - free virus to > 10 rlu at moi  = 10 , overlapping that obtained by capture of hiv-1 t  cells ( figure  5d ) . a similar range of signals was also observed for the t / f virus rejo up to the maximum obtainable moi of 2 . thus , the efficiency of cell - to - cell infection is recapitulated for these viruses by high - moi , cell - free infection . by contrast , despite similar input cell - free moi maxima of 10 for wito and 2 for ch040 , infection levels remained low , giving maximum luciferase values 5- to 50-fold below that obtained by the cell - to - cell route of mdm infection ( figure  5d ) . increasing the moi of cell - free t / f virus inocula therefore increased mdm infection in a virus - strain - dependent manner . since t / f viruses display reduced tropism for mdms compared to chronic m - tropic viruses and may have different sensitivity to inhibitors of cd4- and ccr5-gp120 binding ( chikere et  al . , 2014 ; ochsenbauer et  al . , 2012 ; parker et  al . , 2013 ) , we compared t / f virus inhibition by the same entry inhibitors used against hiv-1bal ( figure  3f ) . mdms were cocultured with autologous t  cells infected with the imc panel , washed , and cultured in the continued presence of the inhibitors for 6  days . all inhibitors strongly inhibited mdm infection by all viruses with the exception of 2g12 , which only weakly inhibited neutralization - resistant strain hiv-1yu2 and some t / f viruses ( figure  5e ) . these data confirm that mdm infection via capture of m - tropic and t / f virus - infected t  cells is equivalently sensitive to entry inhibition . our results reveal a mechanism of efficient hiv-1 cell - to - cell spread driven by mdm capture of hiv-1 t  cells . the finding that macrophage infection may be initiated in this way has implications for infection in  vivo . macrophage recognition and uptake of hiv-1-infected healthy and dying t  cells might take place at all  times during natural infection , and both live and dead / dying t  cells are potent carriers of infectivity ( sealy et  al . , 2009 ) . this mode of viral spread could occur shortly after transmission when t / f viruses replicate locally in lamina propria cd4 t  cells and later when massive infection and death of cd4 t  cells in secondary lymphoid tissues ( li et  al . conversely , the finding that nm - tropic viral gag may persist within t  cells engulfed by macrophages may impact upon interpretation of data from macrophages obtained ex  vivo from hiv- or siv - infected hosts . detection of vdna or viral proteins within phagocytes including macrophages and monocytes may not necessarily represent their infection but may indicate uptake of infected immune cells or their debris . we show that hiv-1-infected t  cells are captured by mdms using interactions apparently distinct from those mediating conventional vs formation ( sattentau , 2008 ) but have yet to define the uptake recognition signals . since immunodeficiency virus - infected t  cells die in  vitro and in  vivo ( li et  al . , 2005 ; mattapallil et  al . , 2005 ) , we explored macrophage recognition of this process by attempting to block death receptor - ligand interactions using published inhibitors . . this might reflect the highly redundant nature of these interactions ( devitt and marshall , 2011 ; poon et  al . , 2010 ) , or that the receptor - ligand interactions implicated in this process are as yet undefined , or that the pathway of macrophage recognition of dead / dying cells is distinct from that used for hiv-1 t  cell recognition . we have not explored the cell biology of hiv-1 infection of macrophages following capture of infected t  cells here . uptake of hiv-1 t  cells by macrophages leading to t  cell degradation in a maturing lysosomal environment may indeed occur for a proportion of engulfed t  cells . however , it is evident from our data that some virus escapes from infected t  cells prior to degradation , leading to robust macrophage infection . whether this takes place during hiv-1 t  cell capture at the plasma membrane or later from within the uptake compartment remains to be determined . regardless of the membrane with which virus fuses , entry via this mode of infection is dependent on the expression of cd4 and ccr5 , as demonstrated by our inhibitor analyses . a somewhat surprising finding was that a small percentage of engulfed t  cells remained intact within macrophages for up to 6  days and possibly longer . although it is unclear whether any of these cells were viable , it appears that they were taken up into a nondegradative compartment within which they persisted for extended periods . the magnitude of mdm infection achieved by capture of t  cells infected by the m - tropic viruses and the t / f virus rejo , which we have previously described as having a stronger macrophage tropism than most t / f viruses ( ochsenbauer et  al . , 2012 ) , was recapitulated by a high cell - free moi infection . thus , for these viruses , the efficiency of this mode of macrophage infection is probably determined solely by quantitative aspects of virus transfer from the infected t  cell . however , even at high moi , two other t / f viruses ( wito and ch040 ) did not achieve a level of infection equivalent to that of cell - to - cell infection , an observation consistent with the particularly weak mdm tropism of these viruses ( ochsenbauer et  al . increasing the cell - free moi to > 10 may overcome entry restrictions to wito and ch040 , giving an equivalent infection to the cell - to - cell route . however , since t / f viruses appear to have a reduced affinity for cd4 and altered interaction with ccr5 compared to m - tropic viruses ( ochsenbauer et  al . 2013 ) , a further possibility is that capture of  hiv-1-infected t  cells by macrophages may drive clustering of viral receptors at the interface , facilitating virus - receptor engagement and entry as reported for vs formation between hiv-1-infected and uninfected t  cells ( jolly et  al . , 2004 ) . the idea that a pathogen may disseminate from one infected cell type to another by phagocyte engulfment is not new and has been termed a strategy with respect to leishmania major parasite dissemination within an infected host ( ritter et  al . , 2009 ) . in this model , neutrophils that engulf l.  major harbor the parasites in an infectious form , ultimately dying and passing them onto their principal host cell , the macrophage ( peters et  al . , similarly , macrophages that have engulfed mycobacterium tuberculosis bacilli undergo apoptosis and are subsequently phagocytosed by newly recruited macrophages , efficiently spreading the infection ( davis and ramakrishnan , 2009 ) . finally , listeria monocytogenes - induced ps exposure on infected cells mimics apoptotic signals that enhance cell - to - cell spread of this bacterium to macrophages ( czuczman et  al . , 2014 ) . our observation of the capture of hiv-1-infected t  cells by macrophages extends this phenomenon to viruses and may have important implications for spread of viruses to phagocytic cells within infected hosts . ( 2008 ) , except that monocytes were negatively selected from donor peripheral blood mononuclear cells using monocyte isolation kit - ii ( miltenyi biotec ) . autologous cd4 t  cells were negatively selected from pha / il-2-stimulated peripheral blood mononuclear cells ( cd4 isolation kit , miltenyi biotec ) and infected with the following viruses : wt hiv-1bal or hiv-1iiib ( nibsc centre for aids reagents , cfar ) , 550  ng ca - p24 per 1   10 cd4 t  cells for 7  days in complete rpmi 1640/10% fetal calf serum/10  u / ml il-2 ( cfar ) . replication - competent imc stably expressing renilla reniformis luciferase in an isogenic nl4.3 backbone ( nl - lucr.t2a ) encoding diverse env ectodomains in cis ( ochsenbauer et  al . , 2012 ) envs were r5  m tropic ( bal , yu2 ) , r5 t / f ( rhpa , thro , wito , rejo , suma and ch040 ) , and x4 nl4.3 nm tropic ( ochsenbauer et  al . , 2012 ) . t  cells were 10%20% gag on the day of use as determined by flow cytometry ( facscalibur , bd biosciences ) . for live - cell imaging experiments , jurkat - tat - r5 or primary cd4 t  cells were infected with the x4 hiv-1nl4.3-gfp or mcherry imc expressing r5 t / f env ch077 ( nl4.3-rfp ( hiv-1ch077/mcherry ) by magnetofection ( sacha and watkins , 2010 ) to achieve > 15% gfp or mcherry cells at 2  days postinfection . t cell to mdm hiv-1 transmission was by direct coculture or across transwell membranes ( 3.0  m , costar ) or cell - free supernatant added directly to target cells . within - donor variability in t  cell infection levels between imcs in mdm infection readouts was normalized in figures 5a and 5b to starting t  cell infection levels . in some experiments , mdms were incubated with azt ( 5  m , cfar ) for up to 12  hr pre - coculture and maintained in the medium . for qpcr assay of vdna , total dna was extracted with the dneasy blood and tissue kit ( qiagen ) and amplified using hiv-1 pol and -globin primers ( martin et  al . , 2010 ) , and results were expressed as pol:-globin to normalize to cell number . for luciferase readout , cell lysates were prepared ( glo - lysis buffer , promega ) and 50  l lysate mixed with 50  l ren - glo luciferase solution ( promega ) at rt and activity ( relative light units , rlu ) measured after 10  min . for gfp - reporting of mdm infection , pha / il-2-activated cd4 t  cells were synchronously magnetofected or mock infected for 48  hr with hiv-1bal - gfp . t  cells were washed , added to autologous mdm 1:1 , and cocultured for 6  hr . following coculture , free or loosely attached t  cells were washed off with 10  mm edta , mdms were cultured , and productive infection was analyzed at day 3 post - coculture by fixing and confocal microscopy . for vsvg - mediated hiv-1 transduction , pha / il-2-activated cd4 t  cells were synchronously magnetofected for 48  hr with hiv-1bal or vsv - g pseudotyped hiv-1balenv ( cfar , nibsc ) or mock infected . infected cd4 t  cells were added to autologous mdm ( 1:1 ) and cocultured for 3  hr . mdms were washed before lifting in 5  mm edta/12  mm lidocaine , fixation , permeabilization , staining , and analysis by flow cytometry . for p24 elisa , supernatants were removed , centrifuged , and inactivated with 0.5% empigen/56c ( groot et  al . , 2008 ) . inhibitors of mdm uptake were jasplakinolide ( 5  m , molecular probes ) and 5-n - ethyl - n - isopropyl amiloride ( eipa , 50  m , sigma - aldrich ) . inhibitors of hiv-1 env - receptor interactions were 13b.8.2 , cd4-specific gp120 blocking mab ( 10  g / ml beckman - coulter ) ; soluble cd4 ( 20  g / ml , iavi ) ; tak-779 ( 500  nm , cfar ) ; 2g12 ( 10  g / ml , iavi ) ; and t20 ( 7.5  g / ml , cfar ) . mdm fc receptors were blocked in 10  g / ml pooled human igg at 37c for 1  hr . inhibitors were preincubated with mdms and/or t  cells at 37c and maintained during coculture ( hiv-1 env receptor inhibitors ) or washed out after preincubation with mdms ( mdm uptake inhibitors ) . cells were fixed in 4% paraformaldehyde ( pfa ) for 1  hr , quenched with 50  mm nh4cl in pbs for 20  min , nuclei stained with 1  g / ml hoechst in pbs , and cells permeabilized in wash buffer ( wb , 0.1% saponin/0.5% bsa in pbs  + 5% pooled human and goat serum ) . samples were stained for cd3 ( ucht1-igg1 , bd biosciences or ucht1-igg2a , p. beverley ) , mouse anti - hiv-1 gag p17 ( cfar , 4c9 ) with anti - mouse alexa 488/647-igg1 or -igg2a ( invitrogen ) . alternatively , directly conjugated mouse anti - hiv-1 gag kc57-fitc ( beckman - coulter ) and phalloidin - tritc ( sigma ) were used . coverslips were mounted in prolonggold antifade ( invitrogen ) and analyzed using an olympus fv1000 . images were acquired with a 60 oil - immersion objective and processed in olympus fluoview v2.0b . autologous hiv-1bal - infected cd4 t  cells were unlabeled or labeled with anti - ps - fitc ( millipore ) , live / dead ( ld)-red ( invitrogen ) . in ps prelabel experiments , cells were postlabeled with cd3-percp cy5.5 ( bd biosciences ) , active caspase3-apc ( bd biosciences ) , and gag ( p17 , p24 , and p55 ) kc57-pe ( beckman coulter ) . for ld experiments or experiments with no t  cell prelabel , cells were labeled with cd3-apc ( bd biosciences ) , caspase3-pe ( bd biosciences ) , and gag kc57-fitc ( beckman coulter ) . mdms and t  cells were cocultured ( 1:1 ) for 3  hr , washed extensively with pbs / edta ( 10  mm ) , and lifted with cold pbs / edta ( 5  mm)/lidocaine ( 12  mm ) . t  cell phenotypes ( figures s1a  s1h ) and t  cell phenotypes associated with mdms ( figures s1i  s1n ) were determined . from this population , 10 mdms per donor were assessed manually , and internalized t  cell phenotypes were scored . the proportions of each t  cell phenotype associated with mdms were determined using the total number of t  cells counted per 10 mdms as the denominator . uptake index equals proportion of t  cell population phenotypes pre - coculture / mdm - associated t  cell phenotypes . for long - term infection experiments , hiv-1bal or hiv-1iiib cd4 t  cells ( 1:1 ) or 6  hr hiv-1bal t  cell supernatants were cocultured with mdm for 6  hr prior to extensive washing . mdms were maintained for 3 to 6  days , lifted , fixed ( as above ) , and stained with cd3-apc and gag kc57-fitc . samples were processed using imagestream 100 or imagestream and analyzed with ideas 4.0 or 5.0 software ( amnis ) . autologous hiv-1bal cd4 t  cells were labeled with ps - fitc , ld - red , or unstained prior to coculture and prepared as for imagestream . antibodies used were as follows : cd14-apc ( biolegend ) , cd14-fitc ( bd biosciences ) , cd3-fitc , cd3-apc , kc57-fitc / pe , cd4-apc , or ccr5-fitc , together with appropriate fluorochrome - conjugated isotype controls . cells were gated according to scatter and staining for cd14 ( mdm ) or cd3 ( t cell ) . all analyses were performed with graphpad prism v5 or v6 , and data were initially interrogated for normal distribution . for group - wise comparisons , one - way anova with bonferonni post hoc test ( parametric ) , kruskal - wallis one - way anova with dunn s post hoc test ( nonparametric ) , or two - way anova with bonferonni post hoc test ( parametric ) were used to account for multiple comparisons . pairwise comparisons were done with student s t test or mann - whitney u. when comparing to a hypothetical mean , one - sampled t tests with sidak s post hoc test correction were used . r.a.r . , and c.j.a.d . conceived , designed , and carried out experimental approaches ; and q.j.s .
summarymacrophages contribute to hiv-1 pathogenesis by forming a viral reservoir and mediating neurological disorders . 
 cell - free hiv-1 infection of macrophages is inefficient , in part due to low plasma membrane expression of viral entry receptors . 
 we find that macrophages selectively capture and engulf hiv-1-infected cd4 + t  cells leading to efficient macrophage infection . 
 infected t  cells , both healthy and dead or  dying , were taken up through viral envelope glycoprotein - receptor - independent interactions , implying a mechanism distinct from conventional virological  synapse formation . 
 macrophages infected by this cell - to - cell route were highly permissive for both ccr5-using macrophage - tropic and otherwise weakly macrophage - tropic transmitted / founder viruses but restrictive for nonmacrophage - tropic cxcr4-using virus . 
 these results have implications for establishment of the macrophage reservoir and hiv-1 dissemination in  vivo .