MicroRNA-433 Inhibits Liver Cancer Cell Migration by Repressing the Protein Expression and Function of cAMP Response Element-binding Protein

We show for the first time that potent microRNA-433 (miR-433) inhibition of expression of the cAMP response element-binding protein CREB1 represses hepatocellular carcinoma (HCC) cell migration. We identified a miR-433 seed match region in human and mouse CREB1 3′-UTRs. Overexpression of miR-433 markedly decreased human CREB1 3′-UTR reporter activity, and the inhibitory effect of miR-433 was alleviated upon mutation of its binding site. Ectopic expression of miR-433 reduced CREB1 protein levels in a variety of human and mouse cancer cells, including HeLa, Hepa1, Huh7, and HepG2. Human CREB1 protein levels in highly invasive MHCC97H cells were diminished by expression of miR-433 but were induced by miR-433 antagomir (anti-miR-433). The expression of mouse CREB1 protein negatively correlated with miR-433 levels in nuclear receptor Shp^−/− liver tissues and liver tumors compared with wild-type mice. miR-433 exhibited a significant repression of MHCC97H cell migration, which was reversed by anti-miR-433. Overexpressing miR-433 inhibited focus formation dramatically, demonstrating that miR-433 may exert a tumor suppressor function. Knockdown of CREB1 by siRNAs impeded MHCC97H cell migration and invasion and antagonized the effect of anti-miR-433. Interestingly, CREB1 siRNA decreased MHCC97H cell proliferation, which was not influenced by anti-miR-433. Overexpressing CREB1 decreased the inhibitory activity of miR-433. The CpG islands surrounding miR-433 were hypermethylated, and the DNA methylation agent 5′-aza-2′-deoxycytidine, but not the histone deacetylase inhibitor trichostatin A, drastically stimulated the expression of miR-433 and miR-127 in HCC cells. The latter is clustered with miR-433. The results reveal a critical role of miR-433 in mediating HCC cell migration via CREB1.     

IL-27 Receptor Signalling Restricts the Formation of Pathogenic,Terminally Differentiated Th1 Cells during Malaria Infection by Repressing IL-12 Dependent Signals

The IL-27R, WSX-1, is required to limit IFN-γ production by effector CD4⁺ T cells in a number of different inflammatory conditions but the molecular basis of WSX-1-mediated regulation of Th1 responses in vivo during infection has not been investigated in detail. In this study we demonstrate that WSX-1 signalling suppresses the development of pathogenic, terminally differentiated (KLRG-1⁺) Th1 cells during malaria infection and establishes a restrictive threshold to constrain the emergent Th1 response. Importantly, we show that WSX-1 regulates cell-intrinsic responsiveness to IL-12 and IL-2, but the fate of the effector CD4⁺ T cell pool during malaria infection is controlled primarily through IL-12 dependent signals. Finally, we show that WSX-1 regulates Th1 cell terminal differentiation during malaria infection through IL-10 and Foxp3 independent mechanisms; the kinetics and magnitude of the Th1 response, and the degree of Th1 cell terminal differentiation, were comparable in WT, IL-10R1^−/− and IL-10^−/− mice and the numbers and phenotype of Foxp3⁺ cells were largely unaltered in WSX-1^−/− mice during infection. As expected, depletion of Foxp3⁺ cells did not enhance Th1 cell polarisation or terminal differentiation during malaria infection. Our results significantly expand our understanding of how IL-27 regulates Th1 responses in vivo during inflammatory conditions and establishes WSX-1 as a critical and non-redundant regulator of the emergent Th1 effector response during malaria infection.     

Dietary Fish Oil Inhibits Pro-Inflammatory and ER Stress Signalling Pathways in the Liver of Sows during Lactation

Lactating sows have been shown to develop typical signs of an inflammatory condition in the liver during the transition from pregnancy to lactation. Hepatic inflammation is considered critical due to the induction of an acute phase response and the activation of stress signaling pathways like the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR), both of which impair animal´s health and performance. Whether ER stress-induced UPR is also activated in the liver of lactating sows and whether dietary fish oil as a source of anti-inflammatory effects n-3 PUFA is able to attenuate hepatic inflammation and ER stress-induced UPR in the liver of sows is currently unknown. Based on this, two experiments with lactating sows were performed. The first experiment revealed that ER stress-induced UPR occurs also in the liver of sows during lactation. This was evident from the up-regulation of a set of genes regulated by the UPR and numerically increased phosphorylation of the ER stress-transducer PERK and PERK-mediated phosphorylation of eIF2α and IκB. The second experiment showed that fish oil inhibits ER stress-induced UPR in the liver of lactating sows. This was demonstrated by decreased mRNA levels of a number of UPR-regulated genes and reduced phosphorylation of PERK and PERK-mediated phosphorylation of eIF2α and IκB in the liver of the fish oil group. The mRNA levels of various nuclear factor-κB-regulated genes encoding inflammatory mediators and acute phase proteins in the liver of lactating sows were also reduced in the fish oil group. In line with this, the plasma levels of acute phase proteins were reduced in the fish oil group, although differences to the control group were not significant. In conclusion, ER stress-induced UPR is present in the liver of lactating sows and fish oil is able to inhibit inflammatory signaling pathways and ER stress-induced UPR in the liver.     

Rac Activation by the T-Cell Receptor Inhibits T Cell Migration

Background

T cell migration is essential for immune responses and inflammation. Activation of the T-cell receptor (TCR) triggers a migration stop signal to facilitate interaction with antigen-presenting cells and cell retention at inflammatory sites, but the mechanisms responsible for this effect are not known.

Methodology/Principal Findings

Migrating T cells are polarized with a lamellipodium at the front and uropod at the rear. Here we show that transient TCR activation induces prolonged inhibition of T-cell migration. TCR pre-activation leads to cells with multiple lamellipodia and lacking a uropod even after removal of the TCR signal. A similar phenotype is induced by expression of constitutively active Rac1, and TCR signaling activates Rac1. TCR signaling acts via Rac to reduce phosphorylation of ezrin/radixin/moesin proteins, which are required for uropod formation, and to increase stathmin phosphorylation, which regulates microtubule stability. T cell polarity and migration is partially restored by inhibiting Rac or by expressing constitutively active moesin.

Conclusions/Significance

We propose that transient TCR signaling induces sustained inhibition of T cell migration via Rac1, increased stathmin phosphorylation and reduced ERM phosphorylation which act together to inhibit T-cell migratory polarity.     

CD8+ T Cell Migration to the Skin Requires CD4+ Help in a Murine Model of Contact Hypersensitivity

The relative roles of CD4+ and CD8+ T cells in contact hypersensitivity responses have not been fully solved, and remain an important question. Using an adoptive transfer model, we investigated the role of the respective T cell subset. Magnetic bead separated CD4+ and CD8+ T cells from oxazolone sensitized C57BL/6 mice were transferred into RAG-/- mice, followed by hapten challenge and analysis of inflammatory parameters at 24 hours post exposure. The CD4+ T cell recipient mice developed partial contact hypersensitivity responses to oxazolone. CD8+ T cells caused significant amplification of the response in recipients of both CD4+ and CD8+ T cells including ear swelling, type 1 inflammatory mediators, and cell killing. Unexpectedly, CD8+ T cells were not sufficient to mediate contact hypersensitivity, although abundantly present in the lymph nodes in the CD8+ T cell reconstituted mice. There were no signs of inflammation at the site of hapten exposure, indicating impaired recruitment of CD8+ T cells in the absence of CD4+ T cells. These data show that CD4+ T cells mediate contact hypersensitivity to oxazolone, but CD8+ T cells contribute with the most potent effector mechanisms. Moreover, our results suggest that CD4+ T cell function is required for the mobilization of CD8+ effector T cells to the site of hapten exposure. The results shed new light on the relative importance of CD4+ and CD8+ T cells during the effector phase of contact hypersensitivity.     

Higher CD27+CD8+ T Cells Percentages during Suppressive Antiretroviral Therapy Predict Greater Subsequent CD4+ T Cell Recovery in Treated HIV Infection

HIV-mediated immune dysfunction may influence CD4⁺ T cell recovery during suppressive antiretroviral therapy (ART). We analyzed cellular biomarkers of immunological inflammation, maturation, and senescence in HIV-infected subjects on early suppressive ART. We performed longitudinal analyses of peripheral immunological biomarkers of subjects on suppressive ART (n = 24) from early treatment (median 6.4 months, interquartile range [IQR] 4.8–13.9 months) to 1–2 years of follow-up (median 19.8 months, IQR 18.3–24.6 months). We performed multivariate regression to determine which biomarkers were associated with and/or predictive of CD4⁺ T cell recovery. After adjusting for the pre-ART CD4⁺ T cell count, age, proximal CD4⁺ T cell count, and length of ART medication, the percentage of CD27⁺CD8⁺ T cells remained significantly associated with the CD4⁺ T cell recovery rate (β = 0.092 cells/ul/month, P = 0.028). In HIV-infected subjects starting suppressive ART, patients with the highest percentage of CD8⁺ T cells expressing CD27 had the greatest rate of CD4⁺ T cell recovery.     

CD28 Promotes CD4+ T Cell Clonal Expansion during Infection Independently of Its YMNM and PYAP Motifs

CD28 is required for maximal proliferation of CD4(+) T cells stimulated through their TCRs. Two sites within the cytoplasmic tail of CD28, a YMNM sequence that recruits PI3K and activates NF-κB and a PYAP sequence that recruits Lck, are candidates as transducers of the signals responsible for these biological effects. We tested this proposition by tracking polyclonal peptide:MHCII-specific CD4(+) T cells in vivo in mice with mutations in these sites. Mice lacking CD28 or its cytoplasmic tail had the same number of naive T cells specific for a peptide:MHCII ligand as wild-type mice. However, the mutant cells produced one tenth as many effector and memory cells as wild-type T cells after infection with bacteria expressing the antigenic peptide. Remarkably, T cells with a mutated PI3K binding site, a mutated PYAP site, or both mutations proliferated to the same extent as wild-type T cells. The only observed defect was that T cells with a mutated PYAP or Y170F site proliferated even more weakly in response to peptide without adjuvant than wild-type T cells. These results show that CD28 enhances T cell proliferation during bacterial infection by signals emanating from undiscovered sites in the cytoplasmic tail.     

Trichinella spiralis Infection Suppressed Gut Inflammation with CD4+CD25+Foxp3+ T Cell Recruitment

In order to know the effect of pre-existing Trichinella spiralis infection on experimentally induced intestinal inflammation and immune responses, we induced colitis in T. spiralis-infected mice and observed the severity of colitis and the levels of Th1, Th2, and regulatory cytokines and recruitment of CD4⁺CD25⁺Foxp3⁺ T (regulatory T; T_reg) cells. Female C57BL/6 mice were infected with 250 muscle larvae; after 4 weeks, induction of experimental colitis was performed using 3% dextran sulfate sodium (DSS). During the induction period, we observed severity of colitis, including weight loss and status of stool, and evaluated the disease activity index (DAI). A significantly low DAI and degree of weight loss were observed in infected mice, compared with uninfected mice. In addition, colon length in infected mice was not contracted, compared with uninfected mice. We also observed a significant increase in production of pro-inflammatory cytokines, IL-6 and IFN-γ, in spleen lymphocytes treated with DSS; however, such an increase was not observed in infected mice treated with DSS. Of particular interest, production of regulatory cytokines, IL-10 and transforming growth factor (TGF)-β, in spleen lymphocytes showed a significant increase in mice infected with T. spiralis. A similar result was observed in mesenteric lymph nodes (MLN). Subsets of the population of T_reg cells in MLN and spleen showed significant increases in mice infected with T. spiralis. In conclusion, T. spiralis infection can inhibit the DSS-induced colitis in mice by enhancing the regulatory cytokine and T_reg cells recruitment.     

Protective Vaccine-Induced CD4+ T Cell-Independent B Cell Responses against Rabies Infection

A major goal in rabies virus (RV) research is to develop a single-dose postexposure prophylaxis (PEP) that would simplify vaccination protocols, reduce costs associated with rabies prevention in humans, and save lives. Live replication-deficient RV-based vaccines are emerging as promising single-dose vaccines to replace currently licensed inactivated RV-based vaccines. Nonetheless, little is known about how effective B cells develop in response to live RV-based vaccination. Understanding this fundamental property of rabies immunology may help in developing a single-dose RV vaccine. Typically, vaccines induce B cells secreting high-affinity, class-switched antibodies during germinal center (GC) reactions; however, there is a lag time between vaccination and the generation of GC B cells. In this report, we show that RV-specific antibodies are detected in mice immunized with live but not inactivated RV-based vaccines before B cells displaying a GC B cell phenotype (B220⁺GL7^hiCD95^hi) are formed, indicating a potential role for T cell-independent and early extrafollicular T cell-dependent antibody responses in the protection against RV infection. Using two mouse models of CD4⁺ T cell deficiency, we show that B cells secreting virus-neutralizing antibodies (VNAs) are induced via T cell-independent mechanisms within 4 days postimmunization with a replication-deficient RV-based vaccine. Importantly, mice that are completely devoid of T cells (B6.129P2-Tcrβ^tm1Mom Tcrδ^tm1Mom/J) show protection against pathogenic challenge shortly after immunization with a live replication-deficient RV-based vaccine. We show that vaccines that can exploit early pathways of B cell activation and development may hold the key for the development of a single-dose RV vaccine wherein the rapid induction of VNA is critical.     

Evolution of CD8+ T Cell Responses after Acute PARV4 Infection

PARV4 is a small DNA human virus that is strongly associated with hepatitis C virus (HCV) and HIV infections. The immunologic control of acute PARV4 infection has not been previously described. We define the acute onset of PARV4 infection and the characteristics of the acute-phase and memory immune responses to PARV4 in a group of HCV- and HIV-negative, active intravenous drug users. Ninety-eight individuals at risk of blood-borne infections were tested for PARV4 IgG. Gamma interferon enzyme-linked immunosorbent spot assays, intracellular cytokine staining, and a tetrameric HLA-A2–peptide complex were used to define the T cell populations responding to PARV4 peptides in those individuals who acquired infection during the study. Thirty-five individuals were found to be PARV4 seropositive at the end of the study, eight of whose baseline samples were found to be seronegative. Persistent and functional T cell responses were detected in the acute infection phase. These responses had an active, mature, and cytotoxic phenotype and were maintained several years after infection. Thus, PARV4 infection is common in individuals exposed to blood-borne infections, independent of their HCV or HIV status. Since PARV4 elicits strong, broad, and persistent T cell responses, understanding of the processes responsible may prove useful for future vaccine design.     

Negative Selection by an Endogenous Retrovirus Promotes a Higher-Avidity CD4+ T Cell Response to Retroviral Infection

Effective T cell responses can decisively influence the outcome of retroviral infection. However, what constitutes protective T cell responses or determines the ability of the host to mount such responses is incompletely understood. Here we studied the requirements for development and induction of CD4⁺ T cells that were essential for immunity to Friend virus (FV) infection of mice, according to their TCR avidity for an FV-derived epitope. We showed that a self peptide, encoded by an endogenous retrovirus, negatively selected a significant fraction of polyclonal FV-specific CD4⁺ T cells and diminished the response to FV infection. Surprisingly, however, CD4⁺ T cell-mediated antiviral activity was fully preserved. Detailed repertoire analysis revealed that clones with low avidity for FV-derived peptides were more cross-reactive with self peptides and were consequently preferentially deleted. Negative selection of low-avidity FV-reactive CD4⁺ T cells was responsible for the dominance of high-avidity clones in the response to FV infection, suggesting that protection against the primary infecting virus was mediated exclusively by high-avidity CD4⁺ T cells. Thus, although negative selection reduced the size and cross-reactivity of the available FV-reactive naïve CD4⁺ T cell repertoire, it increased the overall avidity of the repertoire that responded to infection. These findings demonstrate that self proteins expressed by replication-defective endogenous retroviruses can heavily influence the formation of the TCR repertoire reactive with exogenous retroviruses and determine the avidity of the response to retroviral infection. Given the overabundance of endogenous retroviruses in the human genome, these findings also suggest that endogenous retroviral proteins, presented by products of highly polymorphic HLA alleles, may shape the human TCR repertoire that reacts with exogenous retroviruses or other infecting pathogens, leading to interindividual heterogeneity.     

Functional Exhaustion of CD4+ T Lymphocytes during Primary Cytomegalovirus Infection

Human CMV establishes lifelong persistence after primary infection. Chronic CMV infection is associated with intermittent viral reactivation inducing high frequencies of CD4(+) T lymphocytes with potent antiviral and helper properties. Primary CMV infection is characterized by an intense viral replication lasting for several months. The impact of this prolonged exposure to high Ag loads on the functionality of CD4(+) T cells remains incompletely understood. In pregnant women with primary CMV infection, we observed that CMV-specific CD4(+) T lymphocytes had a decreased capacity to proliferate and to produce IL-2. A very large proportion of CMV-specific CD4(+) T cells had downregulated the expression of CD28, a costimulatory molecule centrally involved in the production of IL-2. Unexpectedly, both CD28(-) and CD28(+)CD4(+) T cells produced low levels of IL-2. This defective production of IL-2 was part of a larger downregulation of cytokine production. Indeed, CMV-specific CD4(+) T cells produced lower amounts of IFN-γ and TNF-α and showed lower functional avidity during primary as compared with chronic infection. Increased programmed death-1 expression was observed in CD28(+) CMV-specific CD4(+) T cells, and programmed death-1 inhibition increased proliferative responses. These results indicate that primary CMV infection is associated with the exhaustion of CMV-specific CD4(+) T cells displaying low functional avidity for viral Ags.     

CD4 T Cell Help Is Limiting and Selective during the Primary B Cell Response to Influenza Virus Infection

Influenza virus vaccination strategies are focused upon the elicitation of protective antibody responses through administration of viral protein through either inactivated virions or live attenuated virus. Often overlooked in this strategy is the CD4 T cell response: how it develops into memory, and how it may support future primary B cell responses to heterologous infection. Through the utilization of a peptide-priming regimen, this study describes a strategy for developing CD4 T cell memory with the capacity to robustly expand in the lung-draining lymph node after live influenza virus infection. Not only were frequencies of antigen-specific CD4 T cells enhanced, but these cells also supported an accelerated primary B cell response to influenza virus-derived protein, evidenced by high anti-nucleoprotein (NP) serum antibody titers early, while there is still active viral replication ongoing in the lung. NP-specific antibody-secreting cells and heightened frequencies of germinal center B cells and follicular T helper cells were also readily detectable in the draining lymph node. Surprisingly, a boosted memory CD4 T cell response was not sufficient to provide intermolecular help for antibody responses. Our study demonstrates that CD4 T cell help is selective and limiting to the primary antibody response to influenza virus infection and that preemptive priming of CD4 T cell help can promote effective and rapid conversion of naive B cells to mature antibody-secreting cells.     

CD4+ T Cell-Derived IL-2 Signals during Early Priming Advances Primary CD8+ T Cell Responses

Stimulating naïve CD8⁺ T cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. In this study, we show that the activation and differentiation of CD8⁺ T cells require IL-2 provided by activated CD4⁺ T cells at the initial priming stage within 0–2.5 hours after stimulation. This critical IL-2 signal from CD4⁺ cells is mediated through the IL-2Rβγ of CD8⁺ cells, which is independent of IL-2Rα. The activation of IL-2 signaling advances the restriction point of the cell cycle, and thereby expedites the entry of antigen-stimulated CD8⁺ T-cell into the S phase. Besides promoting cell proliferation, IL-2 stimulation increases the amount of IFNγ and granzyme B produced by CD8⁺ T cells. Furthermore, IL-2 at priming enhances the ability of P14 effector cells generated by antigen activation to eradicate B16.gp33 tumors in vivo. Therefore, our studies demonstrate that a full CD8⁺ T-cell response is elicited by a critical temporal function of IL-2 released from CD4⁺ T cells, providing mechanistic insights into the regulation of CD8⁺ T cell activation and differentiation.     

Analysis of the Dynamics of Infiltrating CD4+ T Cell Subsets in the Heart during Experimental Trypanosoma cruzi Infection

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects several million people in Latin America. Myocarditis, observed during both the acute and chronic phases of the disease, is characterized by an inflammatory mononuclear cell infiltrate that includes CD4⁺ T cells. It is known that Th1 cytokines help to control infection. The role that T_reg and Th17 cells may play in disease outcome, however, has not been completely elucidated. We performed a comparative study of the dynamics of CD4⁺ T cell subsets after infection with the T. cruzi Y strain during both the acute and chronic phases of the disease using susceptible BALB/c and non-susceptible C57BL/6 mice infected with high or low parasite inocula. During the acute phase, infected C57BL/6 mice showed high levels of CD4⁺ T cell infiltration and expression of Th1 cytokines in the heart associated with the presence of T_reg cells. In contrast, infected BALB/c mice had a high heart parasite burden, low heart CD4⁺ T cell infiltration and low levels of Th1 and inflammatory cytokines, but with an increased presence of Th17 cells. Moreover, an increase in the expression of IL-6 in susceptible mice was associated with lethality upon infection with a high parasite load. Chronically infected BALB/c mice continued to present higher parasite burdens than C57BL/6 mice and also higher levels of IFN-γ, TNF, IL-10 and TGF-β. Thus, the regulation of the Th1 response by T_reg cells in the acute phase may play a protective role in non-susceptible mice irrespective of parasite numbers. On the other hand, Th17 cells may protect susceptible mice at low levels of infection, but could, in association with IL-6, be pathogenic at high parasite loads.     

LFA-1 Regulates CD8+ T Cell Activation via T Cell Receptor-mediated and LFA-1-mediated Erk1/2 Signal Pathways

LFA-1 regulates T cell activation and signal transduction through the immunological synapse. T cell receptor (TCR) stimulation rapidly activates LFA-1, which provides unique LFA-1-dependent signals to promote T cell activation. However, the detailed molecular pathways that regulate these processes and the precise mechanism by which LFA-1 contributes to TCR activation remain unclear. We found LFA-1 directly participates in Erk1/2 signaling upon TCR stimulation in CD8⁺ T cells. The presence of LFA-1, not ligand binding, is required for the TCR-mediated Erk1/2 signal pathway. LFA-1-deficient T cells have defects in sustained Erk1/2 signaling and TCR/CD3 clustering, which subsequently prevents MTOC reorientation, cell cycle progression, and mitosis. LFA-1 regulates the TCR-mediated Erk1/2 signal pathway in the context of immunological synapse for recruitment and amplification of the Erk1/2 signal. In addition, LFA-1 ligation with ICAM-1 generates an additional Erk1/2 signal, which synergizes with the existing TCR-mediated Erk1/2 signal to enhance T cell activation. Thus, LFA-1 contributes to CD8⁺ T cell activation through two distinct signal pathways. We demonstrated that the function of LFA-1 is to enhance TCR signaling through the immunological synapse and deliver distinct signals in CD8⁺ T cell activation.Leukocyte function-associated antigen-1 (LFA-1)² plays an important role in regulating leukocyte adhesion and T cell activation (1, 2). LFA-1 consists of the αL (CD11a) and β2 (CD18) subunits. The ligands for LFA-1 include intercellular adhesion molecular-1 (ICAM-1), ICAM-2, and ICAM-3 (3). LFA-1 participates in the formation of the immunological synapse, which regulates T cell activation synergistically with TCR engagement. The immunological synapse is a specialized structure that forms between the T cell and the APC or target cell (1, 2, 4). The function of the immunological synapse is to facilitate T cell activation and signal transduction. Mice deficient in LFA-1 (CD11a KO) have defects in leukocyte adhesion, lymphocyte proliferation, and tumor rejection (5–7).Upon TCR stimulation, the nascent immunological synapse is initiated with surface receptor clustering and cytoskeleton rearrangement, then followed by mature synapse formation after prolonged stimulation (8, 9). In the mature immunological synapse, LFA-1 forms a ring-like pattern at the peripheral supramolecular activation cluster (pSMAC), which surrounds the central supramolecular activation cluster (cSMAC) containing TCR/CD3/lipid rafts (10, 11). The structure of the mature synapse is stable for hours and thought to be important for sustained TCR signaling (12–14). LFA-1 functions via pSMAC to stabilize the cSMAC and is associated with the induction of T cell proliferation, cytokine production, and lytic granule migration toward cSMAC (1, 15). Although LFA-1-containing pSMAC is self-evident in lipid bilayer systems and cell lines, whether it is required for T cell activation under physiological conditions remains controversial (15).TCR stimulation rapidly induces the functional activity of LFA-1, which then provides unique LFA-1-dependent signals to promote T cell activation (16). The process can be divided into two steps. First, the intracellular signaling from TCR regulating LFA-1 activation is known as “inside-out” signaling; second, activated LFA-1, as a signaling receptor, can feedback to transduce the intracellular signal, the “outside-in” signaling (1, 17). It is widely accepted that TCR stimulation activates LFA-1 through affinity and/or avidity regulation, as supported by increased adhesion to ICAM-1 and pSMAC formation (16, 17). The “inside-out” signal process has been investigated extensively (18–21). The TCR proximal signal molecules, Lck, ZAP-70, and PI3K, are known to be important for TCR signaling to LFA-1 activation (22–26). The molecular mechanisms of LFA-1 “outside-in” signaling have been explored only recently. Perez et al. (27) have demonstrated that LFA-1 and ICAM-1 ligation activates the downstream Erk1/2 MAPK signaling pathway upon TCR stimulation, which ultimately leads to the qualitative modulation of CD4⁺ T cell activation through distinct LFA-1-dependent signals. Another recent study provided compelling evidence that LFA-1 reshapes the Ras MAPK pathway downstream of TCR (28). However, the detailed molecular pathways that regulate these processes are poorly defined. Especially, the evidence in support of a distinctive role for LFA-1 in the T cell signaling pathway has lagged behind; whether the function of LFA-1 is to enhance TCR signaling through the immunological synapse and/or deliver distinct signal in T cell activation and whether LFA-1 is indispensable for or merely assists the existing TCR signal pathway. Furthermore, whether and how TCR proximal signal molecules regulate LFA-1 function remains unknown. Further studies are required to understand the LFA-1 and TCR signaling network.In this study, we found that LFA-1 directly participates in CD8⁺ T cell activation. Upon TCR stimulation, LFA-1 regulates both TCR-mediated and LFA-1-mediated Erk1/2 signal pathways. First, the presence of LFA-1, not ligand binding, is required for the sustained Erk1/2 signaling and TCR/CD3 clustering on the surface of CD8⁺ T cells, subsequently leading to MTOC reorientation, cell cycle progression, and mitosis. Second, LFA-1 ligation with ICAM-1 enhances Erk1/2 signaling, which promotes T cell activation with increased IL-2 production and cell proliferation. This LFA-1-mediated Erk1/2 signal pathway integrates with the existing TCR-mediated Erk1/2 signal pathway to enhance T cell activation.