Activation Associated ERK1/2 Signaling Impairments in CD8+ T Cells Co-Localize with Blunted Polyclonal and HIV-1 Specific Effector Functions in Early Untreated HIV-1 Infection

We recently observed that a large proportion of activated (CD38⁺HLA-DR⁺) CD8⁺ T cells from recently HIV-1-infected adults are refractory to phosphorylation of ERK1/2 kinases (p-ERK1/2-refractory). Given that the ERK1/2 pathway mediates intracellular signaling critical for multiple T cell functions, including key effector functions, the loss of ERK1/2 responsiveness may have broad consequences for CD8⁺ T cell function. In the current study, we hypothesized that the p-ERK1/2-refractory population, localized largely within the activated CD38⁺HLA-DR⁺ CD8⁺ T cell population, would display impairments in CD8⁺ T cell effector functions, such as cytokine production and degranulation, compared to CD8⁺ p-ERK1/2-responsive cells. We further hypothesized that the p-ERK1/2-refractory phenotype is persistent over time during untreated infection, and would correlate with poorer virologic control, in a manner independent of CD8⁺ T cell activation level. We performed single-cell resolution, flow cytometric assays of phospho-kinase responses paired to intracellular cytokine staining in one assay to examine IFN-γ, perforin and CD107α responses in CD8⁺ T cells by ERK1/2 signaling profile. On a per cell basis, p-ERK1/2-refractory cells, which fall predominantly within the activated CD8⁺ T cell compartment, produced less IFN-γ in response to polyclonal or HIV-1 antigen-specific stimulation, and expressed lower levels of perforin and CD107α. The p-ERK1/2 refractory cell population displayed minimal overlap with the PD-1 and Tim-3 inhibitory exhaustion markers and predicted high viral load independent of activation, suggesting that ERK1/2 may be a unique marker and point of intervention for improving CD8⁺ T cell function. Blunted effector functions, secondary to ERK1/2 signaling deficits concentrated within activated CD8⁺ T cells, may contribute to immunodeficiency and underlie the predictive capacity of CD8⁺ T cell activation on HIV-1 disease progression. (270/300).     

Age-Related Expansion of Tim-3 Expressing T Cells in Vertically HIV-1 Infected Children

As perinatally HIV-1-infected children grow into adolescents and young adults, they are increasingly burdened with the long-term consequences of chronic HIV-1 infection, with long-term morbidity due to inadequate immunity. In progressive HIV-1 infection in horizontally infected adults, inflammation, T cell activation, and perturbed T cell differentiation lead to an “immune exhaustion”, with decline in T cell effector functions. T effector cells develop an increased expression of CD57 and loss of CD28, with an increase in co-inhibitory receptors such as PD-1 and Tim-3. Very little is known about HIV-1 induced T cell dysfunction in vertical infection. In two perinatally antiretroviral drug treated HIV-1-infected groups with median ages of 11.2 yr and 18.5 yr, matched for viral load, we found no difference in the proportion of senescent CD28⁻CD57⁺CD8⁺ T cells between the groups. However, the frequency of Tim-3⁺CD8⁺ and Tim-3⁺CD4⁺ exhausted T cells, but not PD-1⁺ T cells, was significantly increased in the adolescents with longer duration of infection compared to the children with shorter duration of HIV-1 infection. PD-1⁺CD8⁺ T cells were directly associated with T cell immune activation in children. The frequency of Tim-3⁺CD8⁺ T cells positively correlated with HIV-1 plasma viral load in the adolescents but not in the children. These data suggest that Tim-3 upregulation was driven by both HIV-1 viral replication and increased age, whereas PD-1 expression is associated with immune activation. These findings also suggest that the Tim-3 immune exhaustion phenotype rather than PD-1 or senescent cells plays an important role in age-related T cell dysfunction in perinatal HIV-1 infection. Targeting Tim-3 may serve as a novel therapeutic approach to improve immune control of virus replication and mitigate age related T cell exhaustion.     

Tim-3-Expressing CD4+ and CD8+ T Cells in Human Tuberculosis (TB) Exhibit Polarized Effector Memory Phenotypes and Stronger Anti-TB Effector Functions

T-cell immune responses modulated by T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) during Mycobacterium tuberculosis (Mtb) infection in humans remain poorly understood. Here, we found that active TB patients exhibited increases in numbers of Tim-3-expressing CD4⁺ and CD8⁺ T cells, which preferentially displayed polarized effector memory phenotypes. Consistent with effector phenotypes, Tim-3⁺CD4⁺ and Tim-3⁺CD8⁺ T-cell subsets showed greater effector functions for producing Th1/Th22 cytokines and CTL effector molecules than Tim-3⁻ counterparts, and Tim-3-expressing T cells more apparently limited intracellular Mtb replication in macrophages. The increased effector functions for Tim-3-expressing T cells consisted with cellular activation signaling as Tim-3⁺CD4⁺ and Tim-3⁺CD8⁺ T-cell subsets expressed much higher levels of phosphorylated signaling molecules p38, stat3, stat5, and Erk1/2 than Tim-3- controls. Mechanistic experiments showed that siRNA silencing of Tim-3 or soluble Tim-3 treatment interfering with membrane Tim-3-ligand interaction reduced de novo production of IFN-γ and TNF-α by Tim-3-expressing T cells. Furthermore, stimulation of Tim-3 signaling pathways by antibody cross-linking of membrane Tim-3 augmented effector function of IFN-γ production by CD4⁺ and CD8⁺ T cells, suggesting that Tim-3 signaling helped to drive stronger effector functions in active TB patients. This study therefore uncovered a previously unknown mechanism for T-cell immune responses regulated by Tim-3, and findings may have implications for potential immune intervention in TB.     

The HIV-1 accessory protein Nef increases surface expression of the checkpoint receptor Tim-3 in infected CD4+ T cells

Prolonged immune activation drives the upregulation of multiple checkpoint receptors on the surface of virus-specific T cells, inducing their exhaustion. Reversing HIV-1-induced T cell exhaustion is imperative for efficient virus clearance; however, viral mediators of checkpoint receptor upregulation remain largely unknown. The enrichment of checkpoint receptors on T cells upon HIV-1 infection severely constrains the generation of an efficient immune response. Herein, we examined the role of HIV-1 Nef in mediating the upregulation of checkpoint receptors on peripheral blood mononuclear cells. We demonstrate that the HIV-1 accessory protein Nef upregulates cell surface levels of the checkpoint receptor T-cell immunoglobulin mucin domain-3 (Tim-3) and that this is dependent on Nef''s dileucine motif LL_164/165. Furthermore, we used a bimolecular fluorescence complementation assay to demonstrate that Nef and Tim-3 form a complex within cells that is abrogated upon mutation of the Nef dileucine motif. We also provide evidence that Nef moderately promotes Tim-3 shedding from the cell surface in a dileucine motif–dependent manner. Treating HIV-1-infected CD4⁺ T cells with a matrix metalloprotease inhibitor enhanced cell surface Tim-3 levels and reduced Tim-3 shedding. Finally, Tim-3-expressing CD4⁺ T cells displayed a higher propensity to release the proinflammatory cytokine interferon-gamma. Collectively, our findings uncover a novel mechanism by which HIV-1 directly increases the levels of a checkpoint receptor on the surface of infected CD4⁺ T cells.     

Latent HIV-1 infection of resting CD4⁺ T cells in the humanized Rag2⁻/⁻ γc⁻/⁻ mouse

Persistent human immunodeficiency virus type 1 (HIV-1) infection of resting CD4⁺ T cells, unaffected by antiretroviral therapy (ART), provides a long-lived reservoir of HIV infection. Therapies that target this viral reservoir are needed to eradicate HIV-1 infection. A small-animal model that recapitulates HIV-1 latency in resting CD4⁺ T cells may accelerate drug discovery and allow the rational design of nonhuman primate (NHP) or human studies. We report that in humanized Rag2^−/− γ_c^−/− (hu-Rag2^−/− γ_c^−/−) mice, as in humans, resting CD4⁺ T cell infection (RCI) can be quantitated in pooled samples of circulating cells and tissue reservoirs (e.g., lymph node, spleen, bone marrow) following HIV-1 infection with the CCR5-tropic JR-CSF strain and suppression of viremia by ART. Replication-competent virus was recovered from pooled resting CD4⁺ T cells in 7 of 16 mice, with a median frequency of 8 (range, 2 to 12) infected cells per million T cells, demonstrating that HIV-1 infection can persist despite ART in the resting CD4⁺ T cell reservoir of hu-Rag2^−/− γ_c^−/− mice. This model will allow rapid preliminary assessments of novel eradication approaches and combinatorial strategies that may be challenging to perform in the NHP model or in humans, as well as a rigorous analysis of the effect of these interventions in specific anatomical compartments.     

PD-1 and Tim-3 Pathways Regulate CD8+ T Cells Function in Atherosclerosis

T cell-mediated immunity plays a significant role in the development of atherosclerosis (AS). There is increasing evidence that CD8⁺ T cells are also involved in AS but their exact roles remain unclear. The inhibitory receptors programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain 3 (Tim-3) are well known inhibitory molecules that play a crucial role in regulating CD8⁺ T cell activation or tolerance. Here, we demonstrate that the co-expression of PD-1 and Tim-3 on CD8⁺ T cells is up-regulated in AS patients. PD-1⁺ Tim-3⁺ CD8⁺ T cells are enriched for within the central T (T_CM) cell subset, with high proliferative activity and CD127 expression. Co-expression of PD-1 and Tim-3 on CD8⁺ T cells is associated with increased anti-atherogenic cytokine production as well as decreased pro-atherogenic cytokine production. Blockade of PD-1 and Tim-3 results in a decrease of anti-atherogenic cytokine production by PD-1⁺ Tim-3⁺ CD8⁺ T cells and in an augmentation of TNF-α and IFN-γ production. These findings highlight the important role of the PD-1 and Tim-3 pathways in regulating CD8⁺ T cells function in human AS.     

Tim-3 Expression Defines Regulatory T Cells in Human Tumors

Tim-3, a member of the novel Tim (T cell immunoglobulin and mucin domain) family, has been reported to negatively regulate the immune responses against viral infection and had implications for autoimmune disease. However, the nature and role of Tim-3⁺ CD4 T cells in human tumors remain largely unknown. In the present study, we characterized Tim-3⁺ CD4 T cells in 100 specimens from human hepatocellular, cervical, colorectal and ovarian carcinoma patients. Compared with peripheral blood and nontumor-infiltrating lymphocytes, the lymphocytes isolated from the corresponding tumor tissues of hepatocellular, cervical, colorectal and ovarian carcinoma patients contained significantly greater proportion of Tim-3⁺ CD4 T cells. The majority of tumor-derived Tim-3⁺ CD4 T cells exhibited an impaired capacity to produce IFN-γ and IL-2, but expressed higher levels of CD25, Foxp3, CTLA-4 and GITR than their Tim-3⁻ CD4 T cell counterparts. In contrast, most Tim-3⁺ CD4 T cells isolated from the paired nontumor tissues and peripheral blood did not express these molecules. Moreover, tumor-derived Tim-3⁺ CD4 T cells, but not tumor-derived Tim-3⁻ CD4 T cells, significantly suppressed the proliferation of autologous CD8⁺ T cells in vitro. Notably, multi-color immunofluorescence and confocal microscopy demonstrated that Tim-3⁺Foxp3⁺CD4⁺ cells were preferentially distributed in the tumor nest rather than the peritumoral stroma of hepatocellular carcinoma. Together, our data indicate that Tim-3-expressing CD4 T cells in human tumors could represent the functional regulatory T cells which contribute to the formation of the immune-suppressive tumor micromilieu.     

Expansion of Monocytic Myeloid-Derived Suppressor Cells Dampens T Cell Function in HIV-1-Seropositive Individuals

T lymphocyte dysfunction contributes to human immunodeficiency virus type 1 (HIV-1) disease progression by impairing antivirus cellular immunity. However, the mechanisms of HIV-1 infection-mediated T cell dysfunction are not completely understood. Here, we provide evidence that expansion of monocytic myeloid-derived suppressor cells (M-MDSCs) suppressed T cell function in HIV-1-infected individuals. We observed a dramatic elevation of M-MDSCs (HLA-DR^−/low CD11b⁺ CD33^+/high CD14⁺ CD15⁻ cells) in the peripheral blood of HIV-1-seropositive subjects (n = 61) compared with healthy controls (n = 51), despite efficacious antiretroviral therapy for nearly 2 years. The elevated M-MDSC frequency in HIV-1⁺ subjects correlated with prognostic HIV-1 disease markers, including the HIV-1 load (r = 0.5957; P < 0.0001), CD4⁺ T cell loss (r = −0.5312; P < 0.0001), and activated T cells (r = 0.4421; P = 0.0004). Functional studies showed that M-MDSCs from HIV-1⁺ subjects suppressed T cell responses in both HIV-1-specific and antigen-nonspecific manners; this effect was dependent on the induction of arginase 1 and required direct cell-cell contact. Further investigations revealed that direct HIV-1 infection or culture with HIV-1-derived Tat protein significantly enhanced human MDSC generation in vitro, and MDSCs from healthy donors could be directly infected by HIV-1 to facilitate HIV-1 replication and transmission, indicating that a positive-feedback loop between HIV-1 infection and MDSC expansion existed. In summary, our studies revealed a novel mechanism of T cell dysfunction in HIV-1-infected individuals and suggested that targeting MDSCs may be a promising strategy for HIV-1 immunotherapy.     

SA-4-1BBL Costimulation Inhibits Conversion of Conventional CD4(+) T Cells into CD4(+)FoxP3(+) T Regulatory Cells by Production of IFN-γ

Tumors convert conventional CD4⁺ T cells into induced CD4⁺CD25⁺FoxP3⁺ T regulatory (iTreg) cells that serve as an effective means of immune evasion. Therefore, the blockade of conventional CD4⁺ T cell conversion into iTreg cells represents an attractive target for improving the efficacy of various immunotherapeutic approaches. Using a novel form of 4-1BBL molecule, SA-4-1BBL, we previously demonstrated that costimulation via 4-1BB receptor renders both CD4⁺and CD8⁺ T effector (Teff) cells refractory to inhibition by Treg cells and increased intratumoral Teff/Treg cell ratio that correlated with therapeutic efficacy in various preclinical tumor models. Building on these studies, we herein show for the first time, to our knowledge, that signaling through 4-1BB inhibits antigen- and TGF-β-driven conversion of naïve CD4⁺FoxP3⁻ T cells into iTreg cells via stimulation of IFN-γ production by CD4⁺FoxP3⁻ T cells. Importantly, treatment with SA-4-1BBL blocked the conversion of CD4⁺FoxP3⁻ T cells into Treg cells by EG.7 tumors. Taken together with our previous studies, these results show that 4-1BB signaling negatively modulate Treg cells by two distinct mechanisms: i) inhibiting the conversion of CD4⁺FoxP3⁻ T cells into iTreg cells and ii) endowing Teff cells refractory to inhibition by Treg cells. Given the dominant role of Treg cells in tumor immune evasion mechanisms, 4-1BB signaling represents an attractive target for favorably tipping the Teff:Treg balance toward Teff cells with important implications for cancer immunotherapy.     

Immune Suppression by Neutrophils in HIV-1 Infection: Role of PD-L1/PD-1 Pathway

HIV-1 infection is associated with a progressive loss of T cell functional capacity and reduced responsiveness to antigenic stimuli. The mechanisms underlying T cell dysfunction in HIV-1/AIDS are not completely understood. Multiple studies have shown that binding of program death ligand 1 (PD-L1) on the surface of monocytes and dendritic cells to PD-1 on T cells negatively regulates T cell function. Here we show that neutrophils in the blood of HIV-1-infected individuals express high levels of PD-L1. PD-L1 is induced by HIV-1 virions, TLR-7/8 ligand, bacterial lipopolysaccharide (LPS), and IFNα. Neutrophil PD-L1 levels correlate with the expression of PD-1 and CD57 on CD4⁺ and CD8⁺ T cells, elevated levels of neutrophil degranulation markers in plasma, and increased frequency of low density neutrophils (LDNs) expressing the phenotype of granulocytic myeloid-derived suppressor cells (G-MDSCs). Neutrophils purified from the blood of HIV-1-infected patients suppress T cell function via several mechanisms including PD-L1/PD-1 interaction and production of reactive oxygen species (ROS). Collectively, the accumulated data suggest that chronic HIV-1 infection results in an induction of immunosuppressive activity of neutrophils characterized by high expression of PD-L1 and an inhibitory effect on T cell function.     

Interleukin 7 Up-regulates CD95 Protein on CD4+ T Cells by Affecting mRNA Alternative Splicing: PRIMING FOR A SYNERGISTIC EFFECT ON HIV-1 RESERVOIR MAINTENANCE*

Interleukin-7 (IL-7) has been used as an immunoregulatory and latency-reversing agent in human immunodeficiency virus type 1 (HIV-1) infection. Although IL-7 can restore circulating CD4⁺ T cell counts in HIV-1-infected patients, the anti-apoptotic and proliferative effects of IL-7 appear to benefit survival and expansion of HIV-1-latently infected memory CD4⁺ T lymphocytes. IL-7 has been shown to elevate CD95 on CD4⁺ T cells in HIV-1-infected individuals and prime CD4⁺ T lymphocytes to CD95-mediated proliferative or apoptotic signals. Here we observed that through increasing microRNA-124, IL-7 down-regulates the splicing regulator polypyrimidine tract binding protein (PTB), leading to inclusion of the transmembrane domain-encoding exon 6 of CD95 mRNA and, subsequently, elevation of CD95 on memory CD4⁺ T cells. Moreover, IL-7 up-regulates cellular FLICE-like inhibitory protein (c-FLIP) and stimulates c-Jun N-terminal kinase (JNK) phosphorylation, which switches CD95 signaling to survival mode in memory CD4⁺ T lymphocytes. As a result, co-stimulation through IL-7/IL-7R and FasL/CD95 signal pathways augments IL-7-mediated survival and expansion of HIV-1-latently infected memory CD4⁺ T lymphocytes. Collectively, we have demonstrated a novel mechanism for IL-7-mediated maintenance of HIV-1 reservoir.     

The Catalytic Activity of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 3 Is Required To Sustain CD4+ CD8+ Thymocyte Survival

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family whose function is largely unknown. Given the central role of MAPKs in T cell development, we hypothesized that ERK3 may regulate thymocyte development. Here we have shown that ERK3 deficiency leads to a 50% reduction in CD4⁺ CD8⁺ (DP) thymocyte number. Analysis of hematopoietic chimeras revealed that the reduction in DP thymocytes is intrinsic to hematopoietic cells. We found that early thymic progenitors seed the Erk3^−/− thymus and can properly differentiate and proliferate to generate DP thymocytes. However, ERK3 deficiency results in a decrease in the DP thymocyte half-life, associated with a higher level of apoptosis. As a consequence, ERK3-deficient DP thymocytes are impaired in their ability to make successful secondary T cell receptor alpha (TCRα) gene rearrangement. Introduction of an already rearranged TCR transgene restores thymic cell number. We further show that knock-in of a catalytically inactive allele of Erk3 fails to rescue the loss of DP thymocytes. Our results uncover a unique role for ERK3, dependent on its kinase activity, during T cell development and show that this atypical MAPK is essential to sustain DP survival during RAG-mediated rearrangements.     

Sprouty2 positively regulates T cell function and airway inflammation through regulation of CSK and LCK kinases

The function of Sprouty2 (Spry2) in T cells is unknown. Using 2 different (inducible and T cell–targeted) knockout mouse strains, we found that Spry2 positively regulated extracellular signal-regulated kinase 1/2 (ERK1/2) signaling by modulating the activity of LCK. Spry2^−/− CD4⁺ T cells were unable to activate LCK, proliferate, differentiate into T helper cells, or produce cytokines. Spry2 deficiency abrogated type 2 inflammation and airway hyperreactivity in a murine model of asthma. Spry2 expression was higher in blood and airway CD4⁺ T cells from patients with asthma, and Spry2 knockdown impaired human T cell proliferation and cytokine production. Spry2 deficiency up-regulated the lipid raft protein caveolin-1, enhanced its interaction with CSK, and increased CSK interaction with LCK, culminating in augmented inhibitory phosphorylation of LCK. Knockdown of CSK or dislodgment of caveolin-1–bound CSK restored ERK1/2 activation in Spry2^−/− T cells, suggesting an essential role for Spry2 in LCK activation and T cell function.

The ubiquitously expressed protein Sprouty2 (Spry2) is a known regulator of Ras/ERK signaling pathway. This study uses in vitro and in vivo models, as well as human specimens, to reveal a mechanism for Spry2 in positively regulating ERK activation in CD4+ T-cells.     

Initiation of Antiretroviral Therapy (ART) at Different Stages of HIV-1 Disease Is Not Associated with the Proportion of Exhausted CD8+ T Cells

CD8⁺ T cell-restricted immunity is important in the control of HIV-1 infection, but continued immune activation results in CD8⁺ T cell dysfunction. Early initiation of antiretroviral treatment (ART) and the duration of ART have been associated with immune reconstitution. Here, we evaluated whether restoration of CD8⁺ T cell function in HIV-1-infected individuals was dependent on early initiation of ART. HIV-specific CD107a, IFNγ, IL-2, TNFα and MIP-1β expression by CD8⁺ T cells and the frequency of CD8⁺ T cells expressing PD-1, 2B4 and CD160 were measured by flow cytometry. The frequency of CD8⁺ T cells expressing the inhibitory markers PD-1, 2B4 and CD160 was lower in ART-treated individuals compared with ART-naïve individuals and similar to the frequency in HIV-uninfected controls. The expression of the three markers was similarly independent of when therapy was initiated. Individuals treated before seroconversion displayed an HIV-specific CD8⁺ T cell response that included all five functional markers; this was not observed in individuals treated after seroconversion or in ART-naïve individuals. In summary, ART appears to restore the total CD8⁺ T cell population to a less exhausted phenotype, independent of the time point of initiation. However, to preserve multifunctional, HIV-1-specific CD8⁺ T cells, ART might have to be initiated before seroconversion.     

Acute Cytomegalovirus Infection Is Associated with Increased Frequencies of Activated and Apoptosis-Vulnerable T Cells in HIV-1-Infected Infants

Cytomegalovirus (CMV) coinfection is associated with infant HIV-1 disease progression and mortality. In a cohort of Kenyan HIV-infected infants, the frequencies of activated (CD38⁺ HLA-DR⁺) and apoptosis-vulnerable (CD95⁺ Bcl-2⁻) CD4⁺ and CD8⁺ T cells increased substantially during acute CMV infection. The frequency of activated CD4⁺ T cells was strongly associated with both concurrent CMV coinfection (P = 0.001) and HIV-1 viral load (P = 0.05). The frequency of apoptosis-vulnerable cells was also associated with CMV coinfection in the CD4 (P = 0.02) and CD8 (P < 0.001) T cell subsets. Similar observations were made in HIV-exposed uninfected infants. CMV-induced increases in T cell activation and apoptosis may contribute to the rapid disease progression in coinfected infants.     

Single mutations in the transmembrane envelope protein abrogate the immunosuppressive property of HIV-1

Background

The outcome of untreated HIV-1 infection is progression to AIDS and death in nearly all cases. Some important exceptions are the small number of patients infected with HIV-1 deleted for the accessory gene, nef. With these infections, disease progression is entirely suppressed or greatly delayed. Whether Nef is critical for high levels of replication or is directly cytotoxic remains controversial. The major problem in determining the role of Nef in HIV/AIDS has been the lack of tractable in vivo models where Nef??s complex pathogenic phenotype can be recapitulated.

Results

Intravenous inoculation (3000 to 600,000 TCIU) of BLT humanized mice with HIV-1_LAI reproducibly establishes a systemic infection. HIV-1_LAI (LAI) replicates to high levels (peak viral load in blood 8,200,000?±?1,800,000 copies of viral RNA/ml, range 3,600,000 to 20,400,000; n?=?9) and exhaustively depletes CD4⁺ T cells in blood and tissues. CD4⁺CD8⁺ thymocytes were also efficiently depleted but CD4⁺CD8^- thymocytes were partially resistant to cell killing by LAI. Infection with a nef-deleted LAI (LAINefdd) gave lower peak viral loads (1,220,000?±?330,000, range 27,000 to 4,240,000; n?=?17). For fourteen of seventeen LAINefdd-infected mice, there was little to no loss of either CD4⁺ T cells or thymocytes. Both LAI- and LAINefdd-infected mice had about 8% of total peripheral blood CD8⁺ T cells that were CD38⁺HLA-DR⁺ compared <1% for uninfected mice. Three exceptional LAINefdd-infected mice that lost CD4⁺ T cells received 600,000 TCIU. All three exhibited peak viral loads over 3,000,000 copies of LAINefdd RNA/ml. Over an extended time course, substantial systemic CD4⁺ T cell loss was observed for the three mice, but there was no loss of CD4⁺CD8⁺ or CD4⁺CD8^- thymocytes.

Conclusion

We conclude Nef is necessary for elevated viral replication and as a result indirectly contributes to CD4⁺ T cell killing. Further, Nef was not necessary for the activation of peripheral blood CD8⁺ T cells following infection. However, CD4⁺CD8⁺ thymocyte killing was dependent on Nef even in cases of elevated LAINefdd replication and T cell loss. This depletion of thymic T cell precursors may be a significant factor in the elevated pathogenicity of CXCR4 trophic HIV-1.     

HIV-1 Clade B Tat, but Not Clade C Tat, Increases X4 HIV-1 Entry into Resting but Not Activated CD4+ T Cells

CXCR4-using human immunodeficiency virus, type 1 (HIV-1) variants emerge late in the course of infection in >40% of individuals infected with clade B HIV-1 but are described less commonly with clade C isolates. Tat is secreted by HIV-1-infected cells where it acts on both uninfected bystander cells and infected cells. In this study, we show that clade B Tat, but not clade C Tat, increases CXCR4 surface expression on resting CD4⁺ T cells through a CCR2b-dependent mechanism that does not involve de novo protein synthesis. The expression of plectin, a cytolinker protein that plays an important role as a scaffolding platform for proteins involved in cellular signaling including CXCR4 signaling and trafficking, was found to be significantly increased following B Tat but not C Tat treatment. Knockdown of plectin using RNA interference showed that plectin is essential for the B Tat-induced translocation of CXCR4 to the surface of resting CD4⁺ T cells. The increased surface CXCR4 expression following B Tat treatment led to increased function of CXCR4 including increased chemoattraction toward CXCR4-using-gp120. Moreover, increased CXCR4 surface expression rendered resting CD4⁺ T cells more permissive to X4 but not R5 HIV-1 infection. However, neither B Tat nor C Tat was able to up-regulate surface expression of CXCR4 on activated CD4⁺ T cells, and both proteins inhibited the infection of activated CD4⁺ T cells with X4 but not R5 HIV-1. Thus, B Tat, but not C Tat, has the capacity to render resting, but not activated, CD4⁺ T cells more susceptible to X4 HIV-1 infection.     

Phenotypic characterization of HIV-specific CD8+ T cells during early and chronic infant HIV-1 infection

Although CD8⁺ T cells play an important role in the containment of adult HIV-1 replication, their role in infant HIV-1 infection is not as well understood. Impaired HIV-specific CD8⁺ T cell responses may underlie the persistently high viral loads observed in infants. We examined the frequency and phenotype of infant HIV-specific CD8⁺ T cells in 7 HIV-infected antiretroviral therapy-naïve infants during the first 2 years of life, using class I HLA tetramers and IFN-γ-ELISPOT. The frequency (0.088–3.9% of CD3⁺CD8⁺ cells) and phenotype (CD27⁺CD28⁻, CD45RA^+/−, CD57^+/−, HLA-DR⁺, CD95⁺) of infant HIV-specific CD8⁺ T cells were similar to reports in adults undergoing early infection. Unlike adults, at 23–24 months post-infection a high frequency of HIV-specific CD8⁺ T cells expressed HLA-DR (mean 80%, range 68–85%) and CD95 (mean 88%, range 79–96%), suggesting sustained activation and vulnerability to apoptosis. Despite comparable expansion of HIV-specific CD8⁺ T cells of a similar phenotype to adults during early infection, infant T cells failed to contain HIV-1 replication, and remained persistently activated and vulnerable to apoptosis during chronic infection.     

The Non-Classical MAP Kinase ERK3 Controls T Cell Activation

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.     

An adenoviral vector encoding dominant negative Cbl lowers the threshold for T cell activation in post-thymic T cells

Cbl family ubiquitin ligases act as key negative regulators of TCR signaling. Knockout mice lacking Cbl-b and c-Cbl show augmented T cell activation and CD28-independent IL-2 production. In order to study Cbl function directly in post-thymic T cells, a DN Cbl adenovirus was generated for transduction of T cells from Coxsackie/adenovirus receptor (CAR) transgenic (Tg) mice. We show that dominant negative (DN) Cbl-transduced CD4⁺ T cells exhibited enhanced IL-2 production upon TCR/CD28 engagement compared with empty adenoviral vector-transduced cells. This augmentation was reflected at both IL-2 mRNA and protein level, and correlated with increased protein phosphorylation of Vav, Akt, ERK, and p38MAPK. Our results indicate that introduction of dominant negative Cbl can potentiate activation of post-thymic CD4⁺ T cells, which argues for development of strategies to interfere with Cbl function as a method of immunopotentiation.