HLA-E–restricted HIV-1–specific CD8+ T cell responses in natural infection

CD8⁺ T cell responses restricted by MHC-E, a nonclassical MHC molecule, have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E–restricted CD8⁺ T cell responses in HIV infection, however, remains unknown. In this study, CD8⁺ T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in vitro assays, we observed HLA-E–restricted T cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immunodominant Gag-KF11 epitope in T cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8⁺ T cell responses. KF11 presented via HLA-E in HIV-infected cells was recognized by antigen-specific CD8⁺ T cells. Importantly, bulk CD8⁺ T cells obtained from HIV-infected individuals recognized infected cells via HLA-E presentation. Ex vivo analyses at the epitope level showed a higher responder frequency of HLA-E–restricted responses to KF11 compared with KL9. Taken together, our findings of HLA-E–restricted HIV-specific immune responses offer intriguing and possibly paradigm-shifting insights into factors that contribute to the immunodominance of CD8⁺ T cell responses in HIV infection.     

Potentiating Functional Antigen-specific CD8+ T Cell Immunity by a Novel PD1 Isoform-based Fusion DNA Vaccine

Understanding and identifying new ways of mounting an effective CD8⁺ T cell immune response is important for eliminating infectious pathogens. Although upregulated programmed death-1 (PD1) in chronic infections (such as HIV-1 and tuberculosis) impedes T cell responses, blocking this PD1/PD-L pathway could functionally rescue the “exhausted” T cells. However, there exists a number of PD1 spliced variants with unknown biological function. Here, we identified a new isoform of human PD1 (Δ42PD1) that contains a 42-nucleotide in-frame deletion located at exon 2 domain found expressed in peripheral blood mononuclear cells (PBMCs). Δ42PD1 appears to function distinctly from PD1, as it does not engage PD-L1/PD-L2 but its recombinant form could induce proinflammatory cytokines. We utilized Δ42PD1 as an intramolecular adjuvant to develop a fusion DNA vaccine with HIV-1 Gag p24 antigen to immunize mice, which elicited a significantly enhanced level of anti-p24 IgG1/IgG2a antibody titers, and important p24-specific and tetramer⁺CD8⁺ T cells responses that lasted for ≥7.5 months. Furthermore, p24-specific CD8⁺ T cells remain functionally improved in proliferative and cytolytic capacities. Importantly, the enhanced antigen-specific immunity protected mice against pathogenic viral challenge and tumor growth. Thus, this newly identified PD1 variant (Δ42PD1) amplifies the generation of antigen-specific CD8⁺ T cell immunity when used in a DNA vaccine.     

IL-15 promotes activation and expansion of CD8+ T cells in HIV-1 infection

In HIV-1–infected patients, increased numbers of circulating CD8⁺ T cells are linked to increased risk of morbidity and mortality. Here, we identified a bystander mechanism that promotes CD8 T cell activation and expansion in untreated HIV-1–infected patients. Compared with healthy controls, untreated HIV-1–infected patients have an increased population of proliferating, granzyme B⁺, CD8⁺ T cells in circulation. Vβ expression and deep sequencing of CDR3 revealed that in untreated HIV-1 infection, cycling memory CD8 T cells possess a broad T cell repertoire that reflects the repertoire of the resting population. This suggests that cycling is driven by bystander activation, rather than specific antigen exposure. Treatment of peripheral blood mononuclear cells with IL-15 induced a cycling, granzyme B⁺ phenotype in CD8⁺ T cells. Moreover, elevated IL-15 expression in the lymph nodes of untreated HIV-1–infected patients correlated with circulating CD8⁺ T cell counts and was normalized in these patients following antiretroviral therapy. Together, these results suggest that IL-15 drives bystander activation of CD8⁺ T cells, which predicts disease progression in untreated HIV-1–infected patients and suggests that elevated IL-15 may also drive CD8⁺ T cell expansion that is linked to increased morbidity and mortality in treated patients.     

Proteome-wide analysis of HIV-specific naive and memory CD4+ T cells in unexposed blood donors

The preexisting HIV-1–specific T cell repertoire must influence both the immunodominance of T cells after infection and immunogenicity of vaccines. We directly compared two methods for measuring the preexisting CD4⁺ T cell repertoire in healthy HIV-1–negative volunteers, the HLA-peptide tetramer enrichment and T cell library technique, and show high concordance (r = 0.989). Using the library technique, we examined whether naive, central memory, and/or effector memory CD4⁺ T cells specific for overlapping peptides spanning the entire HIV-1 proteome were detectable in 10 HLA diverse, HIV-1–unexposed, seronegative donors. HIV-1–specific cells were detected in all donors at a mean of 55 cells/million naive cells and 38.9 and 34.1 cells/million in central and effector memory subsets. Remarkably, peptide mapping showed most epitopes recognized by naive (88%) and memory (56%) CD4⁺ T cells had been previously reported in natural HIV-1 infection. Furthermore, 83% of epitopes identified in preexisting memory subsets shared epitope length matches (8–12 amino acids) with human microbiome proteins, suggestive of a possible cross-reactive mechanism. These results underline the power of a proteome-wide analysis of peptide recognition by human T cells for the identification of dominant antigens and provide a baseline for optimizing HIV-1–specific helper cell responses by vaccination.Only one candidate HIV vaccine, a canarypox vectored gp120 with a protein boost, has shown any efficacy (Rerks-Ngarm et al., 2009). The limited protection correlated with induction of nonneutralizing antibodies to the VI/V2 region of the virus Envelope protein (Env; Rerks-Ngarm et al., 2009; Haynes et al., 2012). This modest success has stimulated efforts to design vaccines that generate more efficient neutralizing antibodies, together with potent CD4⁺ T cell responses capable of providing help to B cells and cytotoxic T cells (Burton et al., 2012). Understanding how the magnitude and specificity of these helper T cells can be optimized will be critical to the design of an effective vaccine.Primary immune responses are probably influenced strongly by the preexisting repertoire of B and T cells. However, characterization and quantification of these repertoires is difficult due to the extremely low number of circulating naive precursor cells (Jenkins et al., 2001; Su et al., 2013). Previous studies of naive CD4⁺ T cell repertoires in humans and mice have relied on magnetic beads to enrich MHC tetramer binding cells (Moon et al., 2007; Kwok et al., 2012; Su et al., 2013). However, although this approach gives precise information on responses to particular MHC-peptide epitopes, it does not measure the total repertoire and misses previously unknown epitopes. An alternative T cell library technique requires no prior knowledge of donor HLA type or epitope specificity (Geiger et al., 2009). The method presorts circulating T cells into naive and memory subsets which are seeded at limiting dilution before polyclonal expansion in the presence of PHA, allogeneic feeder cells, and IL-2. Individual cultures are then screened for proliferative responses to a protein or series of peptides representing the pathogen of interest (Geiger et al., 2009). Combined with epitope mapping and the Poisson distribution, the T cell library technique can provide quantitative data on the specificity of the entire preexisting naive and memory repertoire.The existence of HIV-1–specific memory cells in seronegative donors was originally suggested by studies of highly exposed HIV-1 seronegative (HESN) donors. It has been shown that 25–61% of HESNs have demonstrable HIV-1–specific memory cells, probably primed by exposure to the virus. Surprisingly, HIV-1–specific CD4⁺ T cells were also detected in 24–44% of unexposed donors (Ritchie et al., 2011), although it was not clear whether the latter came from cross-reactive memory T cells or naive T cells primed in vitro. More recently, the existence of low frequency (1–10/million) memory CD4⁺ T cells, specific for a known HIV-1 Gag epitope, was demonstrated by HLA DR4 tetramers in 50% of HIV-1 unexposed HLA DR4⁺ adults (Su et al., 2013), but it was not clear how generalizable the HIV-1 result was beyond the single epitope–HLA DR4 combination.The present study first validates the library technique by direct comparison with the tetramer enrichment method for measuring precursor T cell frequencies. We then use the T cell library technique to provide the first proteome-wide analysis of the frequencies and specificities of preexposure HIV-1–specific naive and memory CD4⁺ T cells in a HLA diverse population of HIV-1 unexposed donors.     

Arming Cytokine-induced Killer Cells With Chimeric Antigen Receptors: CD28 Outperforms Combined CD28–OX40 “Super-stimulation”

Cytokine-induced killer (CIK) cells raised interest for use in cellular antitumor therapy due to their capability to recognize and destroy autologous tumor cells in a HLA-independent fashion. The antitumor attack of CIK cells, predominantly consisting of terminally differentiated CD8⁺CD56⁺ cells, can be improved by redirecting by a chimeric antigen receptor (CAR) that recognizes the tumor cell and triggers CIK cell activation. The requirements for CIK cell activation were, however, so far less explored and are likely to be different from those of “younger” T cells. We revealed that CD28 and OX40 CARs produced higher interferon- secretion as compared with the first-generation ζ-CAR; CD28-ζ and the third-generation CD28-ζ–OX40 CAR, however, performed similar in modulating most CIK cell effector functions. Compared with the CD28-ζ CAR, however, the CD28-ζ–OX40 CAR accelerated terminal maturation of CD56⁺ CIK cells producing high frequencies in activation-induced cell death (AICD) and reduced antitumor efficiency in vivo. Consequently, CD28-ζ CAR CIK cells of CD56⁻ phenotype were superior in redirected tumor cell elimination. CAR-mediated CIK cell activation also increased antigen-independent target cell lysis; the CD28-ζ CAR was more efficient than the CD28-ζ–OX40 CAR. Translated into therapeutic strategies, CAR-redirected CIK cells benefit from CD28 costimulation; “super-costimulation” by the CD28-ζ–OX40 CAR, however, performed less in antitumor efficacy due to increased AICD.     

Dual-Affinity Re-Targeting proteins direct T cell–mediated cytolysis of latently HIV-infected cells

Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell–mediated clearance of HIV-1–infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity–mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4⁺ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8⁺ T cell clearance of CD4⁺ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected–patient resting CD4⁺ T cells. Moreover, DARTs mediated CD8⁺ T cell clearance of HIV from resting CD4⁺ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals.     

HIV-1 Variation Diminishes CD4 T Lymphocyte Recognition

Effective long-term antiviral immunity requires specific cytotoxic T lymphocytes and CD4⁺ T lymphocyte help. Failure of these helper responses can be a principle cause of viral persistence. We sought evidence that variation in HIV-1 CD4⁺ T helper epitopes might contribute to this phenomenon. To determine this, we assayed fresh peripheral blood mononuclear cells from 43 asymptomatic HIV-1⁺ patients for proliferative responses to HIV-1 antigens. 12 (28%) showed a positive response, and we went on to map dominant epitopes in two individuals, to p24 Gag restricted by human histocompatibility leukocyte antigen (HLA)-DR1 and to p17 Gag restricted by HLA-DRB52c. Nine naturally occurring variants of the p24 Gag epitope were found in the proviral DNA of the individual in whom this response was detected. All variants bound to HLA-DR1, but three of these peptides failed to stimulate a CD4⁺ T lymphocyte line which recognized the index sequence. Antigenic variation was also detected in the p17 Gag epitope; a dominant viral variant present in the patient was well recognized by a specific CD4⁺ T lymphocyte line, whereas several natural mutants were not. Importantly, variants detected at both epitopes also failed to stimulate fresh uncultured cells while index peptide stimulated successfully. These results demonstrate that variant antigens arise in HIV-1⁺ patients which fail to stimulate the T cell antigen receptor of HLA class II–restricted lymphocytes, although the peptide epitopes are capable of being presented on the cell surface. In HIV-1 infection, naturally occurring HLA class II–restricted altered peptide ligands that fail to stimulate the circulating T lymphocyte repertoire may curtail helper responses at sites where variant viruses predominate.     

Evidence for a stepwise program of extrathymic T cell development within the human tonsil

The development of a broad repertoire of T cells, which is essential for effective immune function, occurs in the thymus. Although some data suggest that T cell development can occur extrathymically, many researchers remain skeptical that extrathymic T cell development has an important role in generating the T cell repertoire in healthy individuals. However, it may be important in the setting of poor thymic function or congenital deficit and in the context of autoimmunity, cancer, or regenerative medicine. Here, we report evidence that a stepwise program of T cell development occurs within the human tonsil. We identified 5 tonsillar T cell developmental intermediates: (a) CD34⁺CD38^dimLin^– cells, which resemble multipotent progenitors in the bone marrow and thymus; (b) more mature CD34⁺CD38^brightLin^– cells; (c) CD34⁺CD1a⁺CD11c^– cells, which resemble committed T cell lineage precursors in the thymus; (d) CD34^–CD1a⁺CD3^–CD11c^– cells, which resemble CD4⁺CD8⁺ double-positive T cells in the thymus; and (e) CD34^–CD1a⁺CD3⁺CD11c^– cells. The phenotype of each subset closely resembled that of its thymic counterpart. The last 4 populations expressed RAG1 and PTCRA, genes required for TCR rearrangement, and all 5 subsets were capable of ex vivo T cell differentiation. TdT⁺ cells found within the tonsillar fibrous scaffold expressed CD34 and/or CD1a, indicating that this distinct anatomic region contributes to pre–T cell development, as does the subcapsular region of the thymus. Thus, we provide evidence of a role for the human tonsil in a comprehensive program of extrathymic T cell development.     

NLRP3 inflammasome induces CD4+ T cell loss in chronically HIV-1–infected patients

Chronic HIV-1 infection is generally characterized by progressive CD4⁺ T cell depletion due to direct and bystander death that is closely associated with persistent HIV-1 replication and an inflammatory environment in vivo. The mechanisms underlying the loss of CD4⁺ T cells in patients with chronic HIV-1 infection are incompletely understood. In this study, we simultaneously monitored caspase-1 and caspase-3 activation in circulating CD4⁺ T cells, which revealed that pyroptotic and apoptotic CD4⁺ T cells are distinct cell populations with different phenotypic characteristics. Levels of pyroptosis and apoptosis in CD4⁺ T cells were significantly elevated during chronic HIV-1 infection, and decreased following effective antiretroviral therapy. Notably, the occurrence of pyroptosis was further confirmed by elevated gasdermin D activation in lymph nodes of HIV-1–infected individuals. Mechanistically, caspase-1 activation closely correlated with the inflammatory marker expression and was shown to occur through NLRP3 inflammasome activation driven by virus-dependent and/or -independent ROS production, while caspase-3 activation in CD4⁺ T cells was more closely related to T cell activation status. Hence, our findings show that NLRP3-dependent pyroptosis plays an essential role in CD4⁺ T cell loss in HIV-1–infected patients and implicate pyroptosis signaling as a target for anti–HIV-1 treatment.     

Variable HIV peptide stability in human cytosol is critical to epitope presentation and immune escape

Induction of virus-specific CD8⁺ T cell responses is critical for the success of vaccines against chronic viral infections. Despite the large number of potential MHC-I–restricted epitopes located in viral proteins, MHC-I–restricted epitope generation is inefficient, and factors defining the production and presentation of MHC-I–restricted viral epitopes are poorly understood. Here, we have demonstrated that the half-lives of HIV-derived peptides in cytosol from primary human cells were highly variable and sequence dependent, and significantly affected the efficiency of cell recognition by CD8⁺ T cells. Furthermore, multiple clinical isolates of HLA-associated HIV epitope variants displayed reduced half-lives relative to consensus sequence. This decreased cytosolic peptide stability diminished epitope presentation and CTL recognition, illustrating a mechanism of immune escape. Chaperone complexes including Hsp90 and histone deacetylase HDAC6 enhanced peptide stability by transient protection from peptidase degradation. Based on empirical results with 166 peptides, we developed a computational approach utilizing a sequence-based algorithm to estimate the cytosolic stability of antigenic peptides. Our results identify sequence motifs able to alter the amount of peptide available for loading onto MHC-I, suggesting potential new strategies to modulate epitope production from vaccine immunogens.     

PD1-based DNA vaccine amplifies HIV-1 GAG-specific CD8+ T cells in mice

Viral vector–based vaccines that induce protective CD8⁺ T cell immunity can prevent or control pathogenic SIV infections, but issues of preexisting immunity and safety have impeded their implementation in HIV-1. Here, we report the development of what we believe to be a novel antigen-targeting DNA vaccine strategy that exploits the binding of programmed death-1 (PD1) to its ligands expressed on dendritic cells (DCs) by fusing soluble PD1 with HIV-1 GAG p24 antigen. As compared with non–DC-targeting vaccines, intramuscular immunization via electroporation (EP) of the fusion DNA in mice elicited consistently high frequencies of GAG-specific, broadly reactive, polyfunctional, long-lived, and cytotoxic CD8⁺ T cells and robust anti-GAG antibody titers. Vaccination conferred remarkable protection against mucosal challenge with vaccinia GAG viruses. Soluble PD1–based vaccination potentiated CD8⁺ T cell responses by enhancing antigen binding and uptake in DCs and activation in the draining lymph node. It also increased IL-12–producing DCs and engaged antigen cross-presentation when compared with anti-DEC205 antibody-mediated DC targeting. The high frequency of durable and protective GAG-specific CD8⁺ T cell immunity induced by soluble PD1–based vaccination suggests that PD1-based DNA vaccines could potentially be used against HIV-1 and other pathogens.     

Loss of viral fitness and cross-recognition by CD8+ T cells limit HCV escape from a protective HLA-B27–restricted human immune response

There is an association between expression of the MHC class I molecule HLA-B27 and protection following human infection with either HIV or HCV. In both cases, protection has been linked to HLA-B27 presentation of a single immunodominant viral peptide epitope to CD8⁺ T cells. If HIV mutates the HLA-B27–binding anchor of this epitope to escape the protective immune response, the result is a less-fit virus that requires additional compensatory clustered mutations. Here, we sought to determine whether the immunodominant HLA-B27–restricted HCV epitope was similarly constrained by analyzing the replication competence and immunogenicity of different escape mutants. Interestingly, in most HLA-B27–positive patients chronically infected with HCV, the escape mutations spared the HLA-B27–binding anchor. Instead, the escape mutations were clustered at other sites within the epitope and had only a modest impact on replication competence. Further analysis revealed that the cluster of mutations is required for efficient escape because a combination of mutations is needed to impair T cell recognition of the epitope. Artificially introduced mutations at the HLA-B27–binding anchors were found to be either completely cross-reactive or to lead to substantial loss of fitness. These results suggest that protection by HLA-B27 in HCV infection can be explained by the requirement to accumulate a cluster of mutations within the immunodominant epitope to escape T cell recognition.     

Triple bypass: complicated paths to HIV escape

Human immunodeficiency virus (HIV) type 1 is highly efficient at evading immune responses and persisting, ultimately causing fatal immunodeficiency in some patients. Mutation in the epitopes recognized by cytolytic CD8⁺ T cells (CTLs) is one such escape process. A new study now shows that one HIV-1 escape mutation may also result in impaired dendritic cell (DC) activity, possibly impairing later T cell responses to the same and other epitopes. The new data complete our understanding of the mechanisms by which the CTL response to an immunodominant gag epitope presented by human histocompatibility leukocyte antigen (HLA)-B27 is evaded. The complexity of the full escape helps to explain why patients with this HLA type progress to AIDS more slowly than average.     

A participant-derived xenograft model of HIV enables long-term evaluation of autologous immunotherapies

HIV-specific CD8⁺ T cells partially control viral replication and delay disease progression, but they rarely provide lasting protection, largely due to immune escape. Here, we show that engrafting mice with memory CD4⁺ T cells from HIV⁺ donors uniquely allows for the in vivo evaluation of autologous T cell responses while avoiding graft-versus-host disease and the need for human fetal tissues that limit other models. Treating HIV-infected mice with clinically relevant HIV-specific T cell products resulted in substantial reductions in viremia. In vivo activity was significantly enhanced when T cells were engineered with surface-conjugated nanogels carrying an IL-15 superagonist, but it was ultimately limited by the pervasive selection of a diverse array of escape mutations, recapitulating patterns seen in humans. By applying mathematical modeling, we show that the kinetics of the CD8⁺ T cell response have a profound impact on the emergence and persistence of escape mutations. This “participant-derived xenograft” model of HIV provides a powerful tool for studying HIV-specific immunological responses and facilitating the development of effective cell-based therapies.     

Relationship of SARS-CoV-2–specific CD4 response to COVID-19 severity and impact of HIV-1 and tuberculosis coinfection

T cells are involved in control of coronavirus disease 2019 (COVID-19), but limited knowledge is available on the relationship between antigen-specific T cell response and disease severity. Here, we used flow cytometry to assess the magnitude, function, and phenotype of SARS coronavirus 2–specific (SARS-CoV-2–specific) CD4⁺ T cells in 95 hospitalized COVID-19 patients, 38 of them being HIV-1 and/or tuberculosis (TB) coinfected, and 38 non–COVID-19 patients. We showed that SARS-CoV-2–specific CD4⁺ T cell attributes, rather than magnitude, were associated with disease severity, with severe disease being characterized by poor polyfunctional potential, reduced proliferation capacity, and enhanced HLA-DR expression. Moreover, HIV-1 and TB coinfection skewed the SARS-CoV-2 T cell response. HIV-1–mediated CD4⁺ T cell depletion associated with suboptimal T cell and humoral immune responses to SARS-CoV-2, and a decrease in the polyfunctional capacity of SARS-CoV-2–specific CD4⁺ T cells was observed in COVID-19 patients with active TB. Our results also revealed that COVID-19 patients displayed reduced frequency of Mycobacterium tuberculosis–specific CD4⁺ T cells, with possible implications for TB disease progression. These results corroborate the important role of SARS-CoV-2–specific T cells in COVID-19 pathogenesis and support the concept of altered T cell functions in patients with severe disease.     

Primary CTL response magnitude in mice is determined by the extent of naive T cell recruitment and subsequent clonal expansion

CD8⁺ T cell responses to viral infection are characterized by the emergence of dominant and subdominant CTL populations. The immunodominance hierarchies of these populations are highly reproducible for any given spectrum of virus-induced peptide-MHCI complexes and are likely determined by multiple factors. Recent studies demonstrate a direct correlation between naive epitope-specific CD8⁺ T cell precursor (CTLp) frequency and the magnitude of the response after antigen challenge. Thus, the number of available precursors in the naive pool has emerged as a key predictor of immunodominance. In contrast to this, we report here no consistent relationship between CTLp frequency and the subsequent magnitude of the immune response for 4 influenza virus–derived epitopes following intranasal infection of mice with influenza A virus. Rather, the characteristic, antigen-driven T cell immunodominance hierarchy was determined by the extent of recruitment from the available pool of epitope-specific precursors and the duration of their continued expansion over the course of the infection. These findings suggest possibilities for enhancing protective immune memory by maximizing both the size and diversity of typically subdominant T cell responses through rational vaccine design.     

T cell receptor–targeted immunotherapeutics drive selective in vivo HIV- and CMV-specific T cell expansion in humanized mice

To delineate the in vivo role of different costimulatory signals in activating and expanding highly functional virus-specific cytotoxic CD8⁺ T cells, we designed synTacs, infusible biologics that recapitulate antigen-specific T cell activation signals delivered by antigen-presenting cells. We constructed synTacs consisting of dimeric Fc-domain scaffolds linking CD28- or 4-1BB–specific ligands to HLA-A2 MHC molecules covalently tethered to HIV- or CMV-derived peptides. Treatment of HIV-infected donor PBMCs with synTacs bearing HIV- or CMV-derived peptides induced vigorous and selective ex vivo expansion of highly functional HIV- and/or CMV-specific CD8⁺ T cells, respectively, with potent antiviral activities. Intravenous injection of HIV- or CMV-specific synTacs into immunodeficient mice intrasplenically engrafted with donor PBMCs markedly and selectively expanded HIV-specific (32-fold) or CMV-specific (46-fold) human CD8⁺ T cells populating their spleens. Notably, these expanded HIV- or CMV-specific CD8⁺ T cells directed potent in vivo suppression of HIV or CMV infections in the humanized mice, providing strong rationale for consideration of synTac-based approaches as a therapeutic strategy to cure HIV and treat CMV and other viral infections. The synTac platform flexibility supports facile delineation of in vivo effects of different costimulatory signals on patient-derived virus-specific CD8⁺ T cells, enabling optimization of individualized therapies, including HIV cure strategies.