The progressive depletion of CD4 T cells underlies clinical progression to AIDS in untreated HIV-infected subjects. Most dying CD4 T cells correspond to resting nonpermissive cells residing in lymphoid tissues. Death is due to an innate immune response against the incomplete cytosolic viral DNA intermediates accumulating in these cells. The viral DNA is detected by the IFI16 sensor, leading to inflammasome assembly, caspase-1 activation, and the induction of pyroptosis, a highly inflammatory form of programmed cell death. We now show that cell-to-cell transmission of HIV is obligatorily required for activation of this death pathway. Cell-free HIV-1 virions, even when added in large quantities, fail to activate pyroptosis. These findings underscore the infected CD4 T cells as the major killing units promoting progression to AIDS and highlight a previously unappreciated role for the virological synapse in HIV pathogenesis.

Retroviruses disseminate between susceptible cells either by cell-free infection or by direct cell-to-cell spread (). The advantage of cell-to-cell spread on viral infectivity has been recognized for two decades (). For HIV-1, the infectivity of virus-producing cells, as measured in co-culture systems, is ∼10to 10times higher than the infectivity of cell-free particles from the same infected cells (). However, in the context of pathogenesis, it was unclear whether transfer of HIV-1 through cell-to-cell contact triggers the same innate immune responses as cell-free particles in resting CD4 T cells, the predominant target cells depleted by HIV in lymphoid tissues.

The primary cause of AIDS in subjects is the progressive loss of CD4 T cells due to HIV infection (). The depletion of these cells has often been studied using cell-free virions infections of activated blood-derived CD4 T cells because of their ready availability and capacity to support productive viral infection (). However, the cytopathic response to HIV is not restricted to productively infected cells. Indeed, most dying CD4 T cells in lymphoid tissues are resting cells that cannot support productive infection and instead become abortively infected (). We have used an ex vivo human lymphoid aggregate culture (HLAC) system formed with fresh human tonsil tissues to study CD4 T cell death during HIV infection (). HLACs can be infected with a small number of viral particles in the absence of exogenous mitogens, allowing analysis of HIV-1 cytopathicity in a natural and preserved lymphoid microenvironment (). Infection of HLACs with HIV-1 produces extensive loss of CD4 T cells––less than 5% of the cells die as a result of productive viral infection, while >95% of them die as a consequence of abortive infection (). Due to the nonpermissive nature of these quiescent cells, the viral life cycle attenuates during the chain elongation phase of reverse transcription, giving rise to incomplete transcripts of cytosolic viral DNA. These intermediates are sensed by interferon gamma inducible protein 16 (IFI16) (), which activates caspase-1 in inflammasomes, leading in turn to pyroptosis, a highly inflammatory form of programmed cell death ().

Consistent with pyroptosis as the pathway of programmed cell death, spinoculation with multiple-round HIV particles resulted in the release of the intracellular enzyme lactate dehydrogenase (LDH) () ( Figure 4 B). Further, the release of LDH was completely blocked when AMD3100 was added 4 hr after spinoculation or when single-round or integrase-deficient HIV-1 particles were used for initial infection. Together, these findings indicate that infection with cell-free HIV-1 particles does not lead to caspase-1 activation, despite apparent abortive infection of lymphoid CD4 T cells. Rather, capsase-1 activation and the induction of pyroptosis require the generation of productively infected cells and successful cell-to-cell spread of HIV-1 to quiescent bystander lymphoid CD4 T cells.

Most CD4 T cells in lymphoid tissues infected with HIV die by caspase-1-mediated pyroptosis triggered by abortive viral infection (). To test whether caspase-1 is induced by cell-free HIV-1 particles or by cell-to-cell spread of HIV-1, we spinoculated HLACs with single-round or multiple-round clones of a DsRedExpress reporter virus (NLRX-IRES) () and analyzed intracellular caspase-1 activity using cell-permeable fluorogenic caspase-1-specific substrates (CaspaLux1) (). Consistent with our previous reports (), spinoculation with multiple-round HIV-1 particles triggered high levels of intracellular caspase-1 activity in target CD4 T cells. In contrast, spinoculation with single-round or integrase-deficient HIV-1 particles produced only background levels of caspase-1 activity ( Figure 4 A). Inhibition of cell-to-cell spread using the viral protease inhibitor saquinavir, or by treatment with AMD3100 4 hr after spinoculation, also markedly inhibited caspase-1 activation induced by multiple-round HIV-1 particles.

(A) HLACs were spinoculated with multiple- or single-round NLRX-IRES reporter clones as indicated. Saquinavir was added to the culture before spinoculation. AMD3100 was added to the culture 4 hr after spinoculation. Cells were analyzed by flow cytometry using cell-permeable fluorogenic substrates that contain amino acids sequences specifically cleaved by active caspase-1 (CaspaLux1). Abundant caspase-1 activity is exclusively observed in cultures spinoculated with multiple-round HIV-1 clones. Essentially no caspase-1 activity is observed in target CD4 T cells where cell-to-cell spread of HIV-1 is blocked or does not occur. Histograms show one experiment, a representative of three independent experiments performed with tonsils from different donors.

Western blotting analysis of HLAC revealed high expression levels of ICAM-1 in B cells, but not in CD4 or CD8 T lymphocytes. However, activated CD4 T cells, which correspond to those that become productively infected with HIV-1, express high levels of this adhesion molecule ( Figure 3 E). In contrast to ICAM-1, CD11a expression levels were high in both resting and activated CD4 T cells ( Figure 3 F). Thus, synapse formation between activated CD4 T cells expressing ICAM-1 and target CD4 T cells (either activated or resting) expressing LFA-1 may occur regularly in lymphoid tissues, independently of viral infection.

Cell-to-cell spread of HIV-1 predominantly takes place across specialized contact-induced structures known as virological synapses (). These synapses facilitate efficient transmission of virus toward the uninfected and engaged target cell. The synapse gains stability through actin-mediated recruitment of adhesion molecules, such as the integrin leukocyte function-association antigen 1 (LFA-1) and its cognate ligand ICAM-1 to the junction point of cellular interaction (). To examine whether virological synapse formation between HIV-infected and target cells is required to promote CD4 T cell death, productively infected and target CFSE-labeled HLACs were co-cultured in the presence of blocking antibodies against ICAM-1 or CD11a, the α-subunit of the LFA-1 heterodimer. Addition of either the anti-ICAM-1 ( Figure 3 C) or anti-CD11a ( Figure 3 D) antibodies, but not isotype-matched control antibodies, effectively blocked depletion of target CD4 T cells in the mixed cultures as efficiently as the antiviral drug efavirenz. These findings suggest that the death response involves adhesion molecules that are required for virological synapse formation, indicative of a requirement for close cell-cell contact in mediating pyroptotic cell death. Because cell-free virions also express LFA-1 and ICAM-1 (), we cannot completely rule out an additional effect on HIV virions. However, when combined with all of the data ( Figure S2 ), we conclude that cell-to-cell transmission is critical for the induction of pyroptosis.

Presence of host ICAM-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and CD4(+)-T-cell depletion in human lymphoid tissue, a major site of replication in vivo.

To further explore whether cell-cell contact was needed to induce death of CD4 T cells, we repeated the co-culture assay using productively infected and target CFSE-labeled HLACs. However, in this experiment, the cells were co-cultured under conditions of increasing surface area, thereby reducing the likelihood of cell-cell interactions. Using flow cytometry, we analyzed the levels of viable target CD4 T cells in the plates every 24 hr during 4 days of co-culture. The death of target CD4 T cells decreased as the surface area of the culture increased ( Figure 3 A), even in samples where the volume of culture medium remained constant ( Figure 3 B). These data suggest that the physical distance between HIV-producing and target cells directly affects the kinetics of CD4 T cell depletion and argue further against a role for free virions released into the medium in the death response.

(F) High expression of LFA-1 on lymphoid CD4 and CD8 T cells, but not B cells. In contrast to ICAM-1, LFA-1 expression is not increased on activated CD4 T cells.

(B) Inverse correlation between culture surface area and CD4 T cell death. Death of target CD4 T cells in each vessel was examined after 4 days of co-culturing with HIV-infected cells. Note that cell death decreases even in vessels where the volume of culture medium remained constant.

Single-round and integrase-deficient HIV-1 clones are not competent for cell-to-cell dissemination following spinoculation with HLACs. To confirm that the mode of viral transfer influenced the death response of target CD4 T cells, we modified the infection system by overlaying HLACs on a monolayer of 293T cells that had been transfected with these single-round proviral clones ( Figure 2 C). Interestingly, when these single-round viruses were transferred to HLACs by direct interaction with virus-producing 293T cells, a massive killing of target lymphoid CD4 T cells was observed ( Figure 2 D). These results demonstrate that recapitulating the cell-to-cell mode of viral transfer is sufficient to restore the killing capacity of these single-round clones.

Based on these contrasting effects of raltegravir, we hypothesized that in the co-culture experiments involving mixing of HIV-infected HLACs with target cells, raltegravir had no effect on the ensuing death of target CD4 T cells that became abortively infected, because the culture already contained productively infected cells. Conversely, in the spinoculation experiments, raltegravir blocked cell death, because it prevented the establishment of a productively infected subset of cells needed for cell-to-cell spread of the virus to target CD4 T cells. To test this hypothesis, we spinoculated HLACs with either single-round or multiple-round viruses containing a GFP reporter (NLENG1I) (). These viruses permit the dynamics of HIV-1 infection and T cell depletion to be simultaneously monitored in the spinoculated cultures. Four days after spinoculation, we observed a similar number of GFP-positive, productively infected cells with both the single-round and multiple-round viruses, indicating that viral spread was not required to establish an initial population of productively infected cells. However, we observed a massive loss of CD4 T cells only in cultures spinoculated with the multiple-round virus. Notably, spinoculation with an integrase-deficient GFP HIV-1 (NLENG1I-D116N) () resulted in no productive infection and no CD4 T cell death ( Figure 2 A). These results suggested that viral spread from productively infected cells, but not spinoculation of cell-free virions, is promoting the death of non-permissive lymphoid CD4 T cells. In agreement with this conclusion, addition of the AMD3100 entry inhibitor 4 hr after spinoculation efficiently blocked the ensuing death response while not affecting the number of GFP-positive productively infected cells ( Figure 2 B). Moreover, treatment with the viral protease inhibitor saquinavir, which acts during the budding stage of HIV-1 replication, did not inhibit productive infection but prevented CD4 T cell death by newly released HIV-1 virions ( Figure S2 ). These findings further indicated that CD4 T cell death occurs after establishment of productive infection, but not during infection with cell-free viruses.

(D) Single-round and integrase-deficient HIV-1 clones kill target CD4 T cells as efficiently as multiple-round HIV-1 clones when transmitted via virus-producing cells. FACS plots are representative of three independent experiments performed with tonsils from different donors.

(C) A method to assess death of CD4 T cells with non-infectious HIV-1 clones. The single-round and integrase-deficient HIV-1 clones are not competent for multiple rounds of viral replication. Instead, we modified the experimental system by overlaying HLAC cells on a monolayer of 293T cells transfected with these proviral clones, as previously described (). As illustrated, fresh human tonsil is processed into HLAC, and cells are cultured in suspension. After 12 hr, transfected 293T cells in a 24-well plate are washed and overlaid with 4 × 10HLAC cells in RPMI. Virus-producing 293T cells directly interact with target overlaying HLAC cells. After 24–72 hr, HLAC suspensions were collected from wells and analyzed by flow cytometry.

(B) Treatment with the entry inhibitor AMD3100 4 hr after spinoculation with multiple-round NLENG1I HIV-1 particles does not prevent productive infection but efficiently blocks the killing of target resting CD4 T cells. Thus, death of CD4 T cells occurs after establishment of productive infection, but not during initial spinoculation of cell-free viruses. Of note, spinoculation of supernatant from infected HLACs failed to induce death of target CD4 T cells (see Figure S3 ).

(A) HLACs were spinoculated with multiple-round (NLENG1I), single-round (NLENG1-ES-IRES, pseudotyped with HIV-1 Env), or integrase-deficient (NLENG1I-D116N) viral clones containing a GFP reporter. An IRES upstream of the nef gene preserves Nef expression and supports LTR-driven GFP expression in productively infected target cells (). No drugs were added to the spinoculated cultures. The levels of productive infection and CD4 T cell depletion in the cultures were analyzed by flow cytometry 4 days after spinoculation.

To further investigate this surprising result, carboxyfluorescein succinimidyl ester (CFSE)-labeled target CD4 T cells were co-cultured with productively infected HLACs, and raltegravir was added at the time of mixing of productively infected and target CD4 T cells ( Figure 1 C). Under these conditions, raltegravir had no effect on target CD4 T cell death, while efavirenz and AMD3100 blocked the response ( Figure 1 D).

To test this hypothesis, we used spinoculation to emulate efficient cell-to-cell spread of virus (). Spinoculation accelerates the binding of cell-free virions to target cells, facilitates synchronized delivery of a large number of particles into the cells (), and enhances accumulation of cytoplasmic reverse DNA transcripts ( Figure 1 A) (). As expected, spinoculation of HLACs with free HIV-1 promoted high levels of HIV-1 fusion into target lymphoid CD4 T cells ( Figure S1 ). Spinoculation also caused extensive and selective depletion of target CD4 T cells ( Figure 1 B). The relative proportion of CD8 T cells was unaltered. CD3/CD8T cells were similarly depleted, indicating that cell loss was not an artifact of downregulated surface expression of CD4 following direct infection (not shown). Consistent with our previous reports (), loss of CD4 T cells was prevented by addition of efavirenz, an NNRTI that allosterically inhibits HIV-1 reverse transcriptase, and by AMD3100, an entry inhibitor that blocks gp120 engagement of the CXCR4 coreceptor. However, unexpectedly and not in keeping with our previous reports, addition of raltegravir, an integrase inhibitor also blocked CD4 T cell death ( Figures 1 B and S5 ). Because cell death involves viral life cycle events occurring prior to viral integration, raltegravir should act too late to affect the abortive infection process that triggers the pyroptotic response.

The death of lymphoid CD4 T cells was examined by spinoculation of target cells with large quantities of cell-free virions to target cells (A and B), as previously described (), or in co-cultures of CFSE-labeled target CD4 T with productively infected HLACs () (C and D). All samples were infected with a multiple-round X4-tropic NL4-3 strain of HIV-1. NL4-3 was selected because tonsillar tissue contains a high percentage of resting CD4 T cells that express CXCR4 (90%–100%). Target cells were treated with the same concentrations of drugs prior to co-culture with productively infected HLACs or spinoculation with free virions. Inhibitors blocking HIV entry (AMD3100) or early steps of reverse transcription (efavirenz) prevented death of target CD4 T cells. In sharp contrast, inhibiting later events in the viral life cycle (raltegravir) did not prevent cell death in co-cultures with productively infected cells (B) but abrogated the death response of target cells spinoculated with cell-free HIV particles (D). Bar graph represents summary of flow data presented. Error bars represent SD/√n (SEM) of three independent donors. Fluorescence-activated cell sorting (FACS) plots are representative of three independent experiments performed with tonsils from different donors. See also Figure S1

Most studies examining innate immune recognition of HIV-1 have utilized cell-free particles and characterized responses occurring in dendritic cells or macrophages (). More recently, attention has focused on resting CD4 T cells in lymphoid tissues, which are mostly non-permissive for productive HIV infection. We previously have shown that the massive death of lymphoid CD4 T cells that are abortively infected with HIV-1 requires close interaction between uninfected target and HIV-producing cells (). These findings were consistent with in vitro () and in vivo () studies showing that dying non-productively infected cells in human lymph nodes often cluster near productively infected cells (). In contrast, we found that cell-free virions accumulating in the supernatants of HIV-infected HLACs, even at high concentrations, were much less efficient at inducing killing of resting target cells by abortive infection. One potential explanation for these differences was that transfer of cell-free particles may not generate sufficient incomplete reverse DNA transcripts to induce a cytopathic response in target CD4 T cells. Cell-to-cell spread increases infection kinetics by two to three orders of magnitude by directing virus assembly and obviating the rate-limiting step of extracellular diffusion required for cell-free virus to find and engage a susceptible target cell ().

The capsids of HIV-1 and HIV-2 determine immune detection of the viral cDNA by the innate sensor cGAS in dendritic cells.

Discussion