Lymphocyte populations in joint tissues from dogs with inflammatory stifle arthritis and associated degenerative cranial cruciate ligament rupture.

OBJECTIVE: To evaluate lymphocyte populations in stifle synovium and synovial fluid of dogs with degenerative cranial cruciate ligament rupture (CCLR). STUDY DESIGN: Prospective clinical study. ANIMALS: Dogs (n=25) with stifle arthritis and CCLR, 7 dogs with arthritis associated with cartilage degeneration (osteoarthritis [OA]), and 12 healthy Beagle dogs with intact CCL. METHODS: Arthritis was graded radiographically in CCLR dogs. After collection of joint tissues, mononuclear cells were isolated and subsequently analyzed using flow cytometry for expression of CD3, CD4, CD8, and CD21. RESULTS: The proportions of CD4(+) T helper lymphocytes, CD8(+) cytotoxic T lymphocytes, and CD3(+) CD4(-) CD8(-) T lymphocytes were increased in synovium from dogs with CCLR compared with synovium from healthy Beagle dogs (P<.05). The proportion of CD3(+) CD4(-) CD8(-) T lymphocytes in synovial fluid was increased in dogs with CCLR compared with dogs with OA (P<.05). In dogs with CCLR, the proportion of CD3(+) CD4(-) CD8(-) T lymphocytes in synovial fluid was inversely correlated with radiographic arthritis (S(R) =-0.68, P<.005). CONCLUSION: Lymphocytic inflammation of stifle synovium and synovial fluid is an important feature of the CCLR arthropathy. Lymphocyte populations include T lymphocytes expressing CD4 and CD8, and CD3(+) CD4(-) CD8(-) T lymphocytes. Presence of CD3(+) CD4(-) CD8(-) T lymphocytes was associated with development of stifle synovitis. Further work is needed to fully identify the phenotype of these cells.

Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine.

Consistent, high-level, vaccine-induced protection against human malaria has only been achieved by inoculation of Plasmodium falciparum (Pf) sporozoites (SPZ) by mosquito bites. We report that the PfSPZ Vaccine--composed of attenuated, aseptic, purified, cryopreserved PfSPZ--was safe and well tolerated when administered four to six times intravenously (IV) to 40 adults. Zero of six subjects receiving five doses and three of nine subjects receiving four doses of 1.35 × 10(5) PfSPZ Vaccine and five of six nonvaccinated controls developed malaria after controlled human malaria infection (P = 0.015 in the five-dose group and P = 0.028 for overall, both versus controls). PfSPZ-specific antibody and T cell responses were dose-dependent. These data indicate that there is a dose-dependent immunological threshold for establishing high-level protection against malaria that can be achieved with IV administration of a vaccine that is safe and meets regulatory standards.

Poly(I:C) is an effective adjuvant for antibody and multi-functional CD4+ T cell responses to Plasmodium falciparum circumsporozoite protein (CSP) and αDEC-CSP in non human primates.

Development of a fully effective vaccine against the pre-erythrocytic stage of malaria infection will likely require induction of both humoral and cellular immune responses. Protein based vaccines can elicit such broad-based immunity depending on the adjuvant and how the protein is formulated. Here to assess these variables, non human primates (NHP) were immunized three times with Plasmodium falciparum (Pf) circumsporozoite protein (CSP) or CSP cloned into MG38, a monoclonal antibody that targets DEC-205 (αDEC-CSP), an endocytic receptor on dendritic cells (DCs). Both vaccines were administered with or without poly(I:C) as adjuvant. Following three immunizations, the magnitude and quality of cytokine secreting CD4+ T cells were comparable between CSP+poly(I:C) and αDEC-CSP+poly(I:C) groups with both regimens eliciting multi-functional cytokine responses. However, NHP immunized with CSP+poly(I:C) had significantly higher serum titers of CSP-specific IgG antibodies and indirect immunofluorescent antibody (IFA) titers against Pf sporozoites. Furthermore, sera from both CSP or αDEC-CSP+poly(I:C) immunized animals limited sporozoite invasion of a hepatocyte cell line (HC04) in vitro. To determine whether CSP-specific responses could be enhanced, all NHP primed with CSP or αDEC-CSP+poly(I:C) were boosted with a single dose of 150,000 irradiated Pf sporozoites (PfSPZ) intravenously. Remarkably, boosting had no effect on the CSP-specific immunity. Finally, immunization with CSP+poly-ICLC reduced malaria parasite burden in the liver in an experimental mouse model. Taken together, these data showing that poly(I:C) is an effective adjuvant for inducing potent antibody and Th1 immunity with CSP based vaccines offers a potential alternative to the existing protein based pre-erythrocytic vaccines.

Role of direct effects of IFN-gamma on T cells in the regulation of CD8 T cell homeostasis.

It is well recognized that IFN-gamma plays a critical role in the control of CD8 T cell expansion and contraction during immune responses to several intracellular pathogens. However, our understanding of the mechanisms underlying the regulation of T cell fate by IFN-gamma is sorely incomplete. Specifically, it is unclear whether regulation of CD8 T cell homeostasis occurs by a T cell intrinsic IFN-gamma pathway. In this study, we have determined the role of the direct effects of IFN-gamma on T cells in regulating the expansion, contraction, and memory phases of the polyclonal CD8 T cell response to an acute viral infection. Using two complementary approaches we demonstrate that the direct effects of IFN-gamma suppress IL-7R expression on Ag-specific effector CD8 T cells, but clonal expansion or deletion of activated CD8 T cells in vivo can occur in the apparent absence of IFN-gammaR signaling in T cells. These findings have clarified fundamental features of control of T cell homeostasis by IFN-gamma in the context of CD8 T cell memory and protective immunity.

Differential requirement for Lck during primary and memory CD8+ T cell responses.

T cell receptor (TCR) signaling mediates cell fate decisions throughout the life of a T cell. The earliest biochemical events during antigen-stimulated TCR signaling include activation of the Src-family protein tyrosine kinase, p56(Lck) (Lck), which is an integral component of the TCR signaling complex by its association with the cytoplasmic tails of CD8 or CD4. CD8 and Lck are obligatory during thymic selection of CD8+ T cells. What remain unknown are when and with what stringency Lck is required for effective TCR-mediated activation and function throughout the life of a mature CD8+ T cell. Using mice that express an inducible Lck transgene in T cells, we have investigated the temporal importance of Lck-mediated TCR signaling in antigen-specific CD8+ T cell responses during acute viral infections. We show that Lck deficiency induced in naive mice abrogated the antigen-specific activation and clonal expansion of CD8+ T cells during a primary response to acute viral infections. Moreover, the magnitude of primary CD8 T cell expansion depended on the duration of Lck-dependent TCR signaling. Quite unexpectedly, however, Lck was dispensable for enhanced functional avidity, maintenance, and reactivation of memory CD8+ T cells in vitro and in vivo. These observations suggest that the TCR signaling apparatus is rewired from an Lck-dependent state in naive CD8+ T cells to an Lck-independent state in memory CD8+ T cells. Less stringent requirements for antigen-specific TCR signaling to activate memory CD8+ T cells could, in part, account for their unique hyperreactivity to antigen, which contributes to accelerated immune control during secondary infections.

Effect of chronic viral infection on epitope selection, cytokine production, and surface phenotype of CD8 T cells and the role of IFN-gamma receptor in immune regulation.

Regulation of CD8 T cell responses in chronic viral infections is not well understood. In this study, we have compared the CD8 T cell responses to immunodominant and subdominant epitopes during an acute and a chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. The epitope hierarchy of the primary CD8 T cell response was similar in acute and chronic LCMV infections. However, strikingly, the epitope hierarchy of the primary CD8 T cell response was conserved in the T cell memory only in an acute but not in a chronic LCMV infection. Interestingly, in an acute infection, increasing the viral dose caused significant changes in the epitope hierarchy of the LCMV-specific memory CD8 T cell pool, with no effect on the primary CD8 T cell response. Functional and phenotypic analyses revealed that exposure of CD8 T cells to extended periods of antigenic stimulation could lead to long-term defects in cytokine production and alteration in expression of cell surface L-selectin (CD62L). Whereas expression of CD44 was minimally altered, a greater proportion of LCMV-specific memory CD8 T cells were CD62L(low) in mice that have recovered from a chronic LCMV infection, compared with acutely infected mice. Mechanistic studies showed that IFN-gammaR deficiency altered the epitope hierarchy of the pool of LCMV-specific memory CD8 T cells without significantly affecting the immunodominance of the primary CD8 T cell response in an acute infection. Taken together, these findings should further our understanding about the regulation of T cell responses in human chronic viral infections.

Role of cell cycle regulator E2F1 in regulating CD8 T cell responses during acute and chronic viral infection.

To determine the role of cell cycle regulatory protein E2F1 in T cell immunity, we compared antigen-specific CD8 T cell responses between wild type (+/+) and E2F1-deficient (E2F1-/-) mice following an acute and chronic infection with lymphocytic choriomeningitis virus (LCMV). During an acute LCMV infection, although LCMV-specific effector CD8 T cells from E2F1-/- mice were less susceptible to activation-induced cell death (AICD) in vitro, E2F1 deficiency had no significant effect on the: (1) expansion or contraction of virus-specific CD8 T cell responses; (2) proliferative renewal of memory CD8 T cells in both lymphoid and non-lymphoid organs. Importantly, under conditions of repeated antigenic stimulation in the setting of a chronic LCMV infection, E2F1 deficiency did not preclude the exhaustion of CD8 T cells specific to the immunodominant epitope nucleoprotein 396-404 (NP396-404). Taken together, our studies show that E2F1, an important tumor suppressor and cell cycle regulator, may not have a non-redundant role in regulating CD8 T cell responses in acute and chronic LCMV infections.

Dissection of antiviral and immune regulatory functions of tumor necrosis factor receptors in a chronic lymphocytic choriomeningitis virus infection.

The effector function of CD8 T cells is mediated via cell-mediated cytotoxicity and production of cytokines like gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). While the roles of perforin-dependent cytotoxicity, IFN-gamma, and TNF-alpha in controlling acute viral infections are well studied, their relative importance in defense against chronic viral infections is not well understood. Using mice deficient for TNF receptor (TNFR) I and/or II, we show that TNF-TNFR interactions have a dual role in mediating viral clearance and downregulating CD8 and CD4 T-cell responses during a chronic lymphocytic choriomeningitis virus (LCMV) infection. While wild-type (+/+) and TNFR II-deficient (p75(-/-)) mice cleared LCMV from the liver and lung, mice deficient in TNFR I (p55(-/-)) or both TNFR I and TNFR II (double knockout [DKO]) exhibited impaired viral clearance. The inability of p55(-/-) and DKO mice to clear LCMV was not a sequel to either suboptimal activation of virus-specific CD8 or CD4 T cells or impairment in trafficking of LCMV-specific CD8 T cells to the liver and lung. In fact, the expansion of LCMV-specific CD8 and CD4 T cells was significantly higher in DKO mice compared to that in +/+, p55(-/-), and p75(-/-) mice. TNFR deficiency did not preclude the physical deletion of CD8 T cells specific for nucleoprotein 396 to 404 but delayed the contraction of CD8 T-cell responses to the epitopes GP33-41 and GP276-285 in the viral glycoprotein. The antibody response to LCMV was not significantly altered by TNFR deficiency. Taken together, these findings have implications in development of immunotherapy in chronic viral infections of humans.