Brucella abortus induces intracellular retention of MHC‐I molecules in human macrophages down‐modulating cytotoxic CD8+ T cell responses

Brucella abortus elicits a vigorous Th1 immune response which activates cytotoxic T lymphocytes. However, B. abortus persists in its hosts in the presence of CD8⁺ T cells, establishing a chronic infection. Here, we report that B. abortus infection of human monocytes/macrophages inhibited the IFN‐γ‐induced MHC‐I cell surface expression. This phenomenon was dependent on metabolically active viable bacteria. MHC‐I down‐modulation correlated with the development of diminished CD8⁺ cytotoxic T cell response as evidenced by the reduced expression of the activation marker CD107a on CD8⁺ T lymphocytes and a diminished percentage of IFN‐γ‐producing CD8⁺ T cells. Inhibition of MHC‐I expression was not due to changes in protein synthesis. Rather, we observed that upon B. abortus infection MHC‐I molecules were retained within the Golgi apparatus. Overall, these results describe a novel mechanism based on the intracellular sequestration of MHC‐I molecules whereby B. abortus would avoid CD8⁺ cytotoxic T cell responses, evading their immunological surveillance.     

In Vivo Identification and Characterization of CD4+ Cytotoxic T Cells Induced by Virulent Brucella abortus Infection

CD4⁺ T cells display a variety of helper functions necessary for an efficient adaptive immune response against bacterial invaders. This work reports the in vivo identification and characterization of murine cytotoxic CD4⁺ T cells (CD4⁺ CTL) during Brucella abortus infection. These CD4⁺ CTLs express granzyme B and exhibit immunophenotypic features consistent with fully differentiated T cells. They express CD25, CD44, CD62L ,CD43 molecules at their surface and produce IFN-γ. Moreover, these cells express neither the co-stimulatory molecule CD27 nor the memory T cell marker CD127. We show here that CD4⁺ CTLs are capable of cytolytic action against Brucella-infected antigen presenting cells (APC) but not against Mycobacterium-infected APC. Cytotoxic CD4⁺ T cell population appears at early stages of the infection concomitantly with high levels of IFN-γ and granzyme B expression. CD4⁺ CTLs represent a so far uncharacterized immune cell sub-type triggered by early immune responses upon Brucella abortus infection.     

Altered T Lymphocyte Proliferation upon Lipopolysaccharide Challenge Ex Vivo

Context

Sepsis is characterized by the development of adaptive immune cell alterations, which intensity and duration are associated with increased risk of health-care associated infections and mortality. However, pathophysiological mechanisms leading to such lymphocyte dysfunctions are not completely understood, although both intrinsic lymphocyte alterations and antigen-presenting cells (APCs) dysfunctions are most likely involved.

Study

The aim of the current study was to evaluate whether lipopolysaccharide (LPS, mimicking initial Gram negative bacterial challenge) could directly impact lymphocyte function after sepsis. Therefore, we explored ex-vivo the effect of LPS priming on human T lymphocyte proliferation induced by different stimuli.

Results

We showed that LPS priming of PBMCs reduced T cell proliferative response and altered IFNγ secretion after stimulation with OKT3 but not with phytohaemagglutinin or anti-CD2/CD3/CD28-coated beads stimulations. Interestingly only LPS priming of monocytes led to decreased T cell proliferative response as opposed to LPS priming of lymphocytes. Importantly, LPS priming was associated with reduced expression of HLA-DR, CD86 and CD64 on monocytes but not with the modification of CD3, CTLA4, PD-1 and CD28 expressions on lymphocytes. Finally, IFNγ stimulation restored monocytes accessory functions and T cell proliferative response to OKT3.

Conclusion

We conclude that LPS priming does not directly impact lymphocyte functions but reduces APC’s capacity to activate T cells. This recapitulates ex vivo indirect mechanisms participating in sepsis-induced lymphocyte alterations and suggests that monocyte-targeting immunoadjuvant therapies in sepsis may also help to improve adaptive immune dysfunctions. Direct mechanisms impacting lymphocytes being also at play during sepsis, the respective parts of direct versus indirect sepsis-induced lymphocyte alterations remain to be evaluated in clinic.     

CD4+ T Cell-derived IL-10 Promotes Brucella abortus Persistence via Modulation of Macrophage Function

Evasion of host immune responses is a prerequisite for chronic bacterial diseases; however, the underlying mechanisms are not fully understood. Here, we show that the persistent intracellular pathogen Brucella abortus prevents immune activation of macrophages by inducing CD4⁺CD25⁺ T cells to produce the anti-inflammatory cytokine interleukin-10 (IL-10) early during infection. IL-10 receptor (IL-10R) blockage in macrophages resulted in significantly higher NF-kB activation as well as decreased bacterial intracellular survival associated with an inability of B. abortus to escape the late endosome compartment in vitro. Moreover, either a lack of IL-10 production by T cells or a lack of macrophage responsiveness to this cytokine resulted in an increased ability of mice to control B. abortus infection, while inducing elevated production of pro-inflammatory cytokines, which led to severe pathology in liver and spleen of infected mice. Collectively, our results suggest that early IL-10 production by CD25⁺CD4⁺ T cells modulates macrophage function and contributes to an initial balance between pro-inflammatory and anti-inflammatory cytokines that is beneficial to the pathogen, thereby promoting enhanced bacterial survival and persistent infection.     

Association between T lymphocyte sub-sets apoptosis and peripheral blood mononuclear cells oxidative stress in systemic lupus erythematosus

Abstract

Increased oxidative stress and lymphocyte apoptosis are a hallmark of the autoimmune disease systemic lupus erythematosus (SLE). However, the association between oxidative stress and T lymphocytes apoptosis has still to be elucidated in SLE. In order to appraise the interaction between oxidative stress and T lymphocyte apoptosis with the severity of disease, oxidative stress profile and T lymphocytes apoptosis were studied. Increased levels of ROS, MDA and CD4⁺ lymphocyte apoptosis were positively associated with disease activity while decreased levels of GSH and percentage expression of CD4⁺ lymphocyte were negatively associated with disease activity. The decrease in intracellular levels of GSH was negatively associated with T lymphocyte, CD4⁺ lymphocyte, CD8⁺ lymphocyte apoptosis and intracellular caspase-3 expression. The present study suggests that increased T lymphocyte sub-sets apoptosis may be mediated by decreased intracellular glutathione concentration and severity of disease might be enhanced together by over-production of ROS in SLE.     

T-Cell Autophagy Deficiency Increases Mortality and Suppresses Immune Responses after Sepsis

Background

Although the role of autophagy in sepsis has been characterized in several organs, its role in the adaptive immune system remains to be ascertained. This study aimed to investigate the role of autophagy in sepsis-induced T cell apoptosis and immunosuppression, using knockout mice with T cell specific deletion of autophagy essential gene Atg7.

Methods and Results

Sepsis was induced in a cecal ligation and puncture (CLP) model, with T-cell-specific Atg7-knockout mice compared to control mice. Autophagic vacuoles examined by electron microscopy were decreased in the spleen after CLP. Autophagy proteins LC3-II and ATG7, and autophagosomes and autolysosomes stained by Cyto-ID Green and acridine orange were decreased in CD4⁺ and CD8⁺ splenocytes at 18 h and 24 h after CLP. This decrease in autophagy was associated with increased apoptosis of CD4⁺ and CD8⁺ after CLP. Moreover, mice lacking Atg7 in T lymphocytes showed an increase in sepsis-induced mortality, T cell apoptosis and loss of CD4⁺ and CD8⁺ T cells, in comparison to control mice. This was accompanied by suppressed cytokine production of Th1/Th2/Th17 by CD4⁺ T cells, reduced phagocytosis in macrophages and decreased bacterial clearance in the spleen after sepsis.

Conclusion

These results indicated that sepsis led to down-regulation of autophagy in T lymphocytes, which may result in enhanced apoptosis induction and decreased survival in sepsis. Autophagy may therefore play a protective role against sepsis-induced T lymphocyte apoptosis and immunosuppression.     

In Vitro-Stimulated IL-6 Monocyte Secretion and In Vivo Peripheral Blood T Lymphocyte Activation Uniquely Predicted 15-Year Survival in Patients with Head and Neck Squamous Cell Carcinoma

The study was performed in order to determine whether peripheral blood monocyte in vitro function, and lymphocyte in vivo activation at diagnosis, was associated with HPV tumor infection status and 15-year survival in head and neck squamous cell carcinoma (HNSCC) patients. Sixty-five patients from a consecutive cohort of newly diagnosed HNSCCs, together with 18 control patients, were included in the study. Monocyte responsiveness was assessed by measuring monocyte in vitro interleukin (IL)-6 secretions after 24 hours of LPS stimulation in cultures with a serum-free medium. T lymphocyte activation was determined as the fraction of CD71-positive cells on CD3-positive cells by flow cytometry, whereas HPV infection was determined by PCR on formalin-fixed paraffin-embedded (FFPE) tumor tissue. Disease-specific survivals and overall survivals were determined 15 years following inclusion. HPV-positive HNSCC patients had a lower monocyte LPS-stimulated IL-6 response. A high LPS-stimulated monocyte IL-6 response predicted a decreased survival rate (P=0.019). A high percentage of CD71-positive T lymphocytes also predicted an impaired prognosis (P=0.021). The predictive power of IL-6 monocyte LPS-stimulated responses was retained when adjusted for age, gender and TNM stage of the patients. The monocyte and T lymphocyte survival predictions were independent of each other. The survival was particularly low with a combined high activated monocyte and T lymphocyte status. In a multivariate analysis, IL-6 secretion and the percentage of CD71-positive T lymphocytes both uniquely predicted survival independent of HPV infection status. It is postulated that the natural and adaptive immune systems are separately and additionally linked to the clinical aggressiveness of HNSCCs.     

2B4 Is Dispensable for T-Dependent B Cell Immune Responses,but Its Deficiency Leads to Enhanced T-Independent Responses Due to an Increase in Peritoneal Cavity B1b Cells

The signaling lymphocyte activation molecule (SLAM) family plays important roles in adaptive immune responses. Herein, we evaluated whether the SLAM family member 2B4 (CD244) plays a role in immune cell development, homeostasis and antibody responses. We found that the splenic cellularity in Cd244 ^-/- mice was significantly reduced due to a reduction in both CD4 T cells and follicular (Fo) B cells; whereas, the number of peritoneal cavity B cells was increased. These findings led us to examine whether 2B4 modulates B cell immune responses. When we examined T-dependent B cell responses, while there was no difference in the kinetics or magnitude of the antigen-specific IgM and IgG₁ antibody response there was a reduction in bone marrow (BM) memory, but not plasma cells in Cd244 ^-/- mice. When we evaluated T-independent immune responses, we found that antigen-specific IgM and IgG₃ were elevated in the serum following immunization. These data indicate that 2B4 dampens T-independent B cell responses due to a reduction in peritoneal cavity B cells, but has minimal impact on T-dependent B cell responses.     

Lymphocyte traffic through lymph nodes and Peyer's patches of the rat: B- and T-cell-specific migration patterns within the tissue,and their dependence on splenic tissue

The migration routes of lymphocyte subsets through organ compartments are of importance when trying to understand the local events taking place during immune responses. We have therefore studied the traffic of B, T, CD4⁺, and CD8⁺ lymphocytes through lymph nodes and Peyer's patches. At various time points after injection into the rat, labeled lymphocytes were localized, and their phenotype characterized in cryostat sections using immunohistochemistry. Morphometry was also performed, and the recovery of ⁵¹Cr-labeled lymphocytes in these organs was determined. B and T lymphocytes entered the lymph nodes via the high endothelial venules in similar numbers. Most B lymphocytes migrated via the paracortex (T cell area) into the cortex (B cell area), and then back in substantial numbers into the paracortex. In contrast, T lymphocytes predominantly migrated into the paracortex and were rarely seen in the cortex. No obvious differences were seen between various lymph nodes and Peyer's patches and the routes of CD4⁺ and CD8⁺ lymphocytes. After injection of lymphocytes into animals with autotransplanted splenic tissue, the number of B lymphocytes that had migrated into the B cell area of lymph nodes and of Peyer's patches was significantly decreased, whereas CD4⁺ lymphocytes migrated in larger numbers into the T cell area of both organs.This study was supported by the Deutsche Forschungsgemein-schaft (SFB 244, A7).     

Murine and Bovine �æ� T Cells Enhance Innate Immunity against Brucella abortus Infections

γδ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human γδ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of γδ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRδ^−/− mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRγδ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. γδ T cells were the major source of IL-17 following infection and also produced IFN-γ. Depletion of γδ T cells from C57BL/6, IL-17Rα^−/−, and GMCSF^−/− mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when γδ T cells were depleted from IFN-γ^−/− mice, enhanced susceptibility was observed. Neutralization of γδ T cells in the absence of TNF-α did not further impair immunity. In the absence of TNF-α or γδ T cells, B. abortus-infected mice showed enhanced IFN-γ, suggesting that they augmented production to compensate for the loss of γδ T cells and/or TNF-α. While the protective role of γδ T cells was TNF-α-dependent, γδ T cells were not the major source of TNF-α and activation of γδ T cells following B. abortus infection was TNF-α-independent. Additionally, bovine TCRγδ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-γ. Collectively, these results demonstrate γδ T cells are important for early protection to B. abortus infections.     

Suppression of bovine T- and B-lymphocyte responses by fetuin, a bovine glycoprotein

Fetuin, a bovine glycoprotein present in milligram amounts in fetal calf serum, suppressed lymphocyte responses to phytohemagglutinin (PHA) by 70–90%. T-lymphocyte-enriched populations were suppressed by fetuin in their response to PHA indicating direct T-cell regulation and suggesting that macrophages were not required for fetuin activity. Lymphocyte responses to the lipopolysaccharides (LPS) of Escherichia coli and Brucella abortus in the presence of fetuin were markedly reduced compared to cultures without fetuin. Fetuin also suppressed alloantigen recognition in 85% of the mixed-leukocyte responses, and primary in vitro antibody responses induced by the LPS of B. abortus. No toxicity to responding cells or inhibition of early cell division could be demonstrated. A mechanism of regulation appeared to be via suppressor cells. Lymphocytes maintained in fetuin for 48 hr, but not beyond 72 hr, exhibited suppressor cells which could modulate normal adult lymphocyte responses to PHA.     

Selective T cell killing of human lymphocytes by ultraviolet radiation

The effects of ultraviolet radiation (uv) on human B and T lymphocytes were studied. In vitro studies showed that T lymphocytes were more sensitive to uv than B lymphocytes as assessed by eosin-dye exclusion. Following uv exposure, the viable lymphocytes responded to mitogens (PHA, PWM), and functional B lymphocytes were present at a time when no viable T cells were detected. Varying doses of uv were required to abrogate different in vitro responses (proliferative response to antigen or allogeneic cells, MIF production, and cell-mediated lympholysis). In vivo, uv was able to diminish an established cutaneous delayed hypersensitivity response. In vitro uv treatment of parental mouse spleen cells eliminated a graft-versus-host reaction in F₁ recipients as determined by the spleen index. The basis for the differential effect of uv on B and T lymphocyte viability and functional responses is unknown.     

Decursin promotes HIF-1α proteasomal degradation and immune responses in hypoxic tumour microenvironment

BackgroundHypoxia and HIF-1α are important regulators of tumour growth and angiogenesis and could be attractive targets for cancer therapeutics. Decursin is an active compound extracted from the roots of Angelica gigas and has been shown to have potent anti-cancer and anti-angiogenic activities. However, whether decursin regulates HIF-1α activity and immune responses under hypoxic conditions is not yet understood.PurposeThe aim of this study was to identify whether decursin exhibits anti-cancer activity by targeting HIF-1α.Study designWe investigated whether decursin regulates HIF-1α protein stability and increases its degradation. In addition, we determined if decursin increases immune responses in tumour microenvironment to identify its hypoxia-associated anti-cancer activities.Materials and methodsWe performed the hypoxia-responsive element promoter–reporter assay, Western blot analysis, immune-fluorescence assay, semi-quantitative RT-PCR and ELISA for VEGF secretion, CCK-8 assay for cell proliferation, TUNEL assay for apoptosis and invasion assay in A549 human lung cancer or HCT116 human colon cancer cells. In vivo Lewis lung carcinoma (LLC) allograft mouse model was used to check tumour growth and immune responses in tumour microenvironment by immunohistochemistry analysis.ResultsWe observed that decursin inhibited HIF-1 activation under hypoxia by down-regulating the protein level of its subunit HIF-1α. It increased oxygen-dependant hydroxylation and ubiquitination of HIF-1α to promote HIF-1α degradation. Decursin also decreased mRNA expression of HIF-1α target genes. Decursin suppressed cancer cell proliferation, induced apoptosis and inhibited cancer cell invasion under hypoxia in cancer cells. In the allograft mouse tumour model, decursin reduced the hypoxic area and HIF-1α and PD-L1 expression. Infiltrating T cells (CD3+), helper T cells (CD4+) and cytotoxic (CD8+) T cells were accumulated, but regulatory T cells (Foxp3) and myeloid-derived suppressor cell-mediated immune suppressors (Arg1) were attenuated by decursin.ConclusionOur results suggest that decursin is a novel HIF-1α inhibitor that functions by promoting its proteasomal degradation and that it also helps improve T cell activation in tumour microenvironment; these findings provide new explanations about its anti-cancer and anti-angiogenic activity mechanisms.     

Generation and Characterisation of Mice Deficient in the Multi-GTPase Domain Containing Protein,GIMAP8

Background

GTPases of the immunity-associated protein family (GIMAPs) are predominantly expressed in mature lymphocytes. Studies of rodents deficient in GIMAP1, GIMAP4, or GIMAP5 have demonstrated that these GTPases regulate lymphocyte survival. In contrast to the other family members, GIMAP8 contains three potential GTP-binding domains (G-domains), a highly unusual feature suggesting a novel function for this protein. To examine a role for GIMAP8 in lymphocyte biology we examined GIMAP8 expression during lymphocyte development. We also generated a mouse deficient in GIMAP8 and examined lymphocyte development and function.

Principal Findings

We show that GIMAP8 is expressed in the very early and late stages of T cell development in the thymus, at late stages during B cell development, and peripheral T and B cells. We find no defects in T or B lymphocyte development in the absence of GIMAP8. A marginal decrease in the number of recirculating bone marrow B cells suggests that GIMAP8 is important for the survival of mature B cells within the bone marrow niche. We also show that deletion of GIMAP8 results in a delay in apoptotic death of mature T cell in vitro in response to dexamethasone or γ-irradiation. However, despite these findings we find that GIMAP8-deficient mice mount normal primary and secondary responses to a T cell dependent antigen.

Conclusions

Despite its unique structure, GIMAP8 is not required for lymphocyte development but appears to have a minor role in maintaining recirculating B cells in the bone marrow niche and a role in regulating apoptosis of mature T cells.     

Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220+ Double-Negative T Lymphocytes of Autoimmune MRL/lpr Mice

Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including Fas Ligand (FasL) and P2X7 receptor (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/lpr mice (lpr mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220⁺ CD4^–CD8^– (DN) T lymphocytes. The precise ontogeny of B220⁺ DN T cells is unknown. B220⁺ DN T lymphocytes could be derived from effector CD4⁺ and CD8⁺ T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/lpr mice carry a P2X7R allele, which confers a high sensitivity to ATP. However, during aging, the MRL/lpr T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220⁺ DN T-cell numbers in lymphoid organs. Importantly, we found that this B220⁺ DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220⁺ T cells observed in normal MRL^+/+ and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220⁺ T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.     

NKp46+ Innate Lymphoid Cells Dampen Vaginal CD8 T Cell Responses following Local Immunization with a Cholera Toxin-Based Vaccine

Innate and adaptive immune cells work in concert to generate efficient protection at mucosal surface. Vaginal mucosa is an epithelial tissue that contains innate and adaptive immune effector cells. Our previous studies demonstrated that vaginal administration of Cholera toxin -based vaccines generate antigen-specific CD8 T cells through the stimulation of local dendritic cells (DC). Innate lymphoid cells (ILC) are a group of lymphocytes localized in epithelial tissues that have important immune functions against pathogens and in tissue homeostasis. Their contribution to vaccine-induced mucosal T cell responses is an important issue for the design of protective vaccines. We report here that the vaginal mucosa contains a heterogeneous population of NKp46⁺ ILC that includes conventional NK cells and ILC1-like cells. We show that vaginal NKp46⁺ ILC dampen vaccine-induced CD8 T cell responses generated after local immunization. Indeed, in vivo depletion of NKp46⁺ ILC with anti-NK1.1 antibody or NKG2D blockade increases the magnitude of vaginal OVA-specific CD8 T cells. Furthermore, such treatments also increase the number of DC in the vagina. NKG2D ligands being expressed by vaginal DC but not by CD8 T cells, these results support that NKp46⁺ ILC limit mucosal CD8 T cell responses indirectly through the NKG2D-dependent elimination of vaginal DC. Our data reveal an unappreciated role of NKp46⁺ ILC in the regulation of mucosal CD8 T cell responses.     

SOCS5 is expressed in primary B and T lymphoid cells but is dispensable for lymphocyte production and function

Suppressors of cytokine signaling (SOCSs) are key regulators of cytokine-induced responses in hematopoietic as well as nonhematopoietic cells. SOCS1 and SOCS3 have been shown to modulate T-cell responses, whereas the roles of other SOCS family members in the regulation of lymphocyte function are less clear. Here, we report the generation of mice with a targeted disruption of the Socs5 gene. Socs5^−/− mice were born in a normal Mendelian ratio and were healthy and fertile. We found that SOCS5 is expressed in primary B and T cells in wild-type mice. However, no abnormalities in the lymphocyte compartment were seen in SOCS5-deficient mice. We examined antigen- and cytokine-induced proliferative responses in B and T cells in the absence of SOCS5 and found no deviations from the responses seen in wild-type cells. Because SOCS5 has been implicated in Th1 differentiation, we also investigated the importance of SOCS5 in T helper cell responses. Unexpectedly, SOCS5-deficient CD4 T cells showed no abnormalities in Th1/Th2 differentiation and Socs5^−/− mice showed normal resistance to infection with Leishmania major. Therefore, although SOCS5 is expressed in primary B and T cells, it appears to be dispensable for the regulation of lymphocyte function.     

Functional CD8+ T cell responses in lethal Ebola virus infection

Ebola virus (EBOV) causes highly lethal hemorrhagic fever that leads to death in up to 90% of infected humans. Like many other infections, EBOV induces massive lymphocyte apoptosis, which is thought to prevent the development of a functional adaptive immune response. In a lethal mouse model of EBOV infection, we show that there is an increase in expression of the activation/maturation marker CD44 in CD4(+) and CD8(+) T cells late in infection, preceding a dramatic rebound of lymphocyte numbers in the blood. Furthermore, we observed both lymphoblasts and apoptotic lymphocytes in spleen late in infection, suggesting that there is lymphocyte activation despite substantial bystander apoptosis. To test whether these activated lymphocytes were functional, we performed adoptive transfer studies. Whole splenocytes from moribund day 7 EBOV-infected animals protected naive animals from EBOV, but not Marburgvirus, challenge. In addition, we observed EBOV-specific CD8(+) T cell IFN-gamma responses in moribund day 7 EBOV-infected mice, and adoptive transfer of CD8(+) T cells alone from day 7 mice could confer protection to EBOV-challenged naive mice. Furthermore, CD8(+) cells from day 7, but not day 0, mice proliferated after transfer to infected recipients. Therefore, despite significant lymphocyte apoptosis, a functional and specific, albeit insufficient, adaptive immune response is made in lethal EBOV infection and is protective upon transfer to naive infected recipients. These findings should cause a change in the current view of the 'impaired' immune response to EBOV challenge and may help spark new therapeutic strategies to control lethal filovirus disease.