Comparing the relative role of perforin/granzyme versus Fas/Fas ligand cytotoxic pathways in CD8+ T cell-mediated insulin-dependent diabetes mellitus.

CD8+ cytotoxic T cells play a critical role in initiating insulin-dependent diabetes mellitus. The relative contribution of each of the major cytotoxic pathways, perforin/granzyme and Fas/Fas ligand (FasL), in the induction of autoimmune diabetes remains controversial. To evaluate the role of each lytic pathway in beta cell lysis and induction of diabetes, we have used a transgenic mouse model in which beta cells expressing the influenza virus hemagglutinin (HA) are destroyed by HA-specific CD8+ T cells from clone-4 TCR-transgenic mice. Upon adoptive transfer of CD8+ T cells from perforin-deficient clone-4 TCR mice, there was a 30-fold increase in the number of T cells required to induce diabetes. In contrast, elimination of the Fas/FasL pathway of cytotoxicity had little consequence. When both pathways of cytolysis were eliminated, mice did not become diabetic. Using a model of spontaneous diabetes, which occurs in double transgenic neonates that express both clone-4 TCR and Ins-HA transgenes, mice deficient in either the perforin or FasL/Fas lytic pathway become diabetic soon after birth. This indicates that, in the neonate, large numbers of autoreactive CD8+ T cells can lead to destruction of islet beta cells by either pathway.     

The role of adoptively transferred CD8 T cells and host cells in the control of the growth of the EG7 thymoma: factors that determine the relative effectiveness and homing properties of Tc1 and Tc2 effectors

We had previously examined the factors that regulate the response of OVA-specific TCR-transgenic CD8 T cells to the B16 OVA melanoma, growing as lung metastases. We examine here whether the same parameters operate for EG7, growing intradermally. Tc1 or Tc2 CD8 effector cells from OT-1 mice were injected either mixed with the tumor or i.v. at day 0 or 7. Tc2 were one-fifth to one-tenth as effective as Tc1 when injected with the tumor, in controlling tumor growth, but were only 1/20 to 1/100 injected i.v. Tc1 injected i.v. entered the draining lymph nodes faster than Tc2 and caused a faster accumulation of host cells. Both caused an abrupt termination of host cell entry into lymph nodes and spleen after tumor elimination, but this occurred earlier for Tc1 than for Tc2. Host responses were ineffective in the absence of adoptive transfer but were essential after transfer. Perforin expression in the donor cells plays no role in adoptively transferred Tc1 or Tc2 control of the tumor, and neither IL-4 nor IL5 is needed for Tc1 or Tc2 function. Tc1 cells from mice lacking IFN-gamma, however, control tumor growth less well, whereas Tc2 effectors lacking IFN-gamma are unaffected. Tc1 from IFN-gamma-deficient mice attract fewer host cells to the draining lymph node, whereas Tc1 cells from perforin-deficient donors are unimpaired. We conclude that host cell recruitment is a crucial element in adoptive immunotherapy. The differences between the EG7 and the previous B16 melanoma model are discussed.     

CD8(+) T cells secreting type 2 lymphokines are defective in protection against viral infection

Effector T cells secreting type 1 and/or type 2 lymphokines (Tc1, Tc0, Tc2) were generated in vitro from CD8(+) T cells of mice with a transgenic TCR recognizing lymphocytic choriomeningitis virus (LCMV) glycoprotein to compare their effector function in vitro and in vivo. Tc1, Tc2, and Tc0 showed similar Fas- and perforin-mediated cytotoxicity in vitro. Upon adoptive transfer, Tc2 and Tc0 effectors were less efficient than Tc1 at controlling LCMV or recombinant vaccinia virus expressing the LCMV glycoprotein in vivo. Tc2 and Tc0 had decreased surface VLA-4 density and deficient activation-induced LFA-1/ICAM-1-dependent homotypic adhesion in vitro. Therefore, the reduced antiviral activity in vivo of Tc2 and Tc0 compared with Tc1 is not due to reduced cytotoxic activity or IFN-gamma secretion but may be explained by defective homing to the target organ due to decreased expression and/or lower activity of adhesion molecules.     

CD8-mediated type 1 antitumor responses selectively modulate endogenous differentiated and nondifferentiated T cell localization, activation, and function in progressive breast cancer

CD8 T cell-mediated immune responses fall into two distinct types based on effector cell-derived cytokine production. Type I CD8 T cells (Tc1) produce IFN-gamma, whereas type 2 cells (Tc2) secrete IL-4, IL-5, IL-10, and GM-CSF. Using a murine TCR transgenic T cell/breast tumor model, we show that adoptively transferred Ag-specific Tc1 cells are more effective in delaying mammary tumor growth and progression than that of functionally distinct Tc2 cells. Donor Tc1 cells administered 7 days posttumor challenge localized and persisted at sites of primary tumor growth with antitumor responses that were dependent, in part, on effector cell-derived IFN-gamma. Tc1-mediated responses markedly enhanced the appearance and local accumulation of highly differentiated (CD44(high)) CD4 and CD8 endogenous tumor-infiltrating T cells when compared with that of untreated tumor-bearing mice. Conversely, Tc1 cell transfer markedly delayed the appearance of corresponding nondifferentiated (CD44(low)) endogenous T cells. Such cells were acutely activated as defined by coexpression of surface markers associated with TCR engagement (CD69) and early T cell activation (CD25). Moreover, cellular response kinetics appeared to further correlate with the up-regulation of endogenous T cells producing the chemokine IFN-gamma-inducible protein-10 in vivo. This suggested that CD8-mediated type 1 antitumor responses cannot only promote accumulation of distinct endogenous CD4 and CD8 T cell subpopulations, but also facilitate and preferentially modulate their localization kinetics, persistence, states of activation/differentiation, and function within the primary tumor environment at various stages of tumor progression. These studies offer insight into potential mechanisms for enhancing T cell-based immunotherapy in breast cancer.     

An essential contribution by IFN-gamma to CD8+ T cell-mediated rejection of pancreatic islet allografts

CD8+ T cells have long been considered to be the prototypical cytotoxic lymphocyte subpopulation. However, whether alloreactive CD8+ T cells require traditional cytolytic pathways such as perforin and Fas ligand (FasL) to mediate graft rejection has been a controversial issue. In the present studies, we examined the role of varied effector pathways in CD8+ T cell-mediated rejection of pancreatic islet allografts. Our goal was to systematically determine the relative requirements, if any, of perforin and FasL as well as the proinflammatory cytokine IFN-gamma in triggering graft destruction. To study CD8+ T cell effector pathways independently of other lymphocyte populations, purified alloreactive CD8+ T cells were adoptively transferred into severe combined immune-deficient (SCID) recipients bearing established islet allografts. Results indicate that to reject established islet allografts, primed CD8+ T cells do not require the individual action of the conventional cytotoxic effectors perforin and Fas ligand. In contrast, the ability to produce IFN-gamma is critical for efficient CD8+ T cell-mediated rejection of established islet allografts. Furthermore, alloreactive CD8+ TCR transgenic T cells (2C) also show IFN-gamma dependence for mediating islet allograft rejection in vivo. We speculate from these results that the production of IFN-gamma by alloreactive CD8+ T cells is a rate-limiting step in the process of islet allograft rejection.     

Role of effector cell-derived IL-4, IL-5, and perforin in early and late stages of type 2 CD8 effector cell-mediated tumor rejection.

Type 2 CD8 T cells (Tc2) secrete IL-4 and IL-5 and display perforin-dependent cytolysis in vitro. Using an OVA-transfected B16-melanoma model, we show that tumor-reactive Tc2 effector cells accumulated at the tumor site and induced tumor regression that enhanced survival in mice with pulmonary tumors. Transfer of perforin-deficient Tc2 cells generated from perforin gene knockout mice showed no differences in therapeutic efficiency when compared with wild-type Tc2 cells. In contrast, Tc2 cells derived from select cytokine gene-deficient mice showed that therapeutic effects were dependent on effector cell-derived IL-4 and IL-5 that led to a local elevation in lung-derived chemoattractants and accumulation of activated host-derived CD8/CD44(high), CD4/CD44(high), and OVA-specific tetramer-positive CD8 cells in vivo. Host-derived T and non-T immune cells increased in the lung over time and correlated with an elevated production of type 1-related chemokines. Conversely, donor Tc2 cell numbers markedly diminished at later times, suggesting that prolonged therapeutic responses were due to host-derived mechanisms. Moreover, type 1 host responses were detectable with increased levels of IFN-gamma production by lung-derived CD4 and CD8 T cells from surviving Tc2-treated mice. Transfer of Tc2 cells into IFN-gamma-deficient tumor-bearing mice was markedly less effective then into wild-type mice, suggesting that host-derived IFN-gamma-dependent mechanisms play a role in Tc2-mediated antitumor responses.     

Alternative mechanisms of respiratory syncytial virus clearance in perforin knockout mice lead to enhanced disease

Virus-specific cytotoxic T lymphocytes are key effectors for the clearance of virus-infected cells and are required for the normal clearance of respiratory syncytial virus (RSV) in mice. Although perforin/granzyme-mediated lysis of infected cells is thought to be the major molecular mechanism used by CD8(+) cytotoxic T lymphocytes for elimination of virus, its role in RSV has not been reported. Here, we show that viral clearance in perforin knockout (PKO) mice is slightly delayed but that both PKO and wild-type mice clear virus by day 10, suggesting an alternative mechanism of RSV clearance. Effector T cells from the lungs of both groups of mice were shown to lyse Fas (CD95)-overexpressing target cells in greater numbers than target cells expressing low levels of Fas, suggesting that Fas ligand (CD95L)-mediated target cell lysis was occurring in vivo. This cell lysis was associated with a delay in RSV-induced disease in PKO mice compared to the time of disease onset for wild-type controls, which correlated with increased and prolonged production of gamma interferon and tumor necrosis factor alpha levels in PKO mice. We conclude that while perforin is not necessary for the clearance of primary RSV infection, the use of alternative CTL target cell killing mechanisms is less efficient and can lead to enhanced disease.     

CD95 ligand-expressing tumors are rejected in anti-tumor TCR transgenic perforin knockout mice

CD95 (APO-/Fas) ligand (CD95L) is a member of the TNF family predominantly expressed by activated T and NK cells but also by tumors of diverse cellular origin. CD95L trimerizes surface CD95 expressed by target cells that subsequently undergo apoptosis. The role of the CD95/CD95L system in the down-regulation of an immune response (activation-induced cell death) is established. However, it is so far unclear why tumors express CD95L. To investigate whether tumors use the CD95L to down-regulate an anti-tumor immune response, we established a transgenic (tg) mouse model consisting of 1) apoptosis-resistant tumor cells, designated LKC-CD95L, which express functional CD95L and the model tumor Ag K(b); and 2) perforin knockout (PKO) anti-K(b) TCR tg mice. L1210-Fas antisense expressing K(b), crmA, and CD95L (LKC-CD95L) killed CD95(+) unrelated tumor targets and Con A-activated splenocytes from anti-K(b) TCR tg PKO mice by a CD95L-dependent mechanism in vitro. However, we could not detect any cytotoxic activity against anti-tumor (anti-K(b)) T cells in vivo. We also observed reduced growth of LKC-CD95L in nude mice and rapid rejection in anti-K(b) TCR tg PKO mice. Because the tumor cells are resistant to CD95L-, TNF-alpha-, and TNF-related apoptosis-inducing ligand-induced apoptosis and the mice used are perforin-deficient, the involvement of these four cytotoxicity mechanisms in tumor rejection can be excluded. The histological examination of tumors grown in nude mice showed infiltration of LKC-CD95L tumors by neutrophils, whereas L1210-Fas antisense expressing K(b) and crmA (LKC) tumor tissue was neutrophil-free. Chemotaxis experiments revealed that CD95L has no direct neutrophil-attractive activity. Therefore, we conclude that LKC-CD95L cells used an indirect mechanism to attract neutrophils that may cause tumor rejection.     

Cytotoxic T cells specifically induce Fas on target cells, thereby facilitating exocytosis-independent induction of apoptosis

Cytotoxic T (Tc) cells deficient in perforin lyse Fas-negative targets after lengthy incubation periods. This process is independent of granzymes, and killing occurs via the Fas pathway for the following reasons. Interaction of perforin-deficient Tc cells with Fas-negative targets leads to an up-regulation of Fas that is dependent on Ag recognition, de novo synthesis, and transport of proteins to the target cell surface. Treatment of effectors with brefeldin A, but not with the exocytosis inhibitor concanamycin, inhibited this process. Lysis of targets is inhibited by anti-Fas Abs, soluble mouse Fas-Fc, and the caspase-cascade inhibitor, crm-A. Targets from Fas-mutant lpr mice are refractory to lysis, and Tc cells from mice deficient in Fas- and perforin-mediated lysis do not lyse Fas-negative targets. The possible relevance of this exocytosis-independent cytolytic process in the regulation of T cell activity and control of pathogens is discussed.     

Liver damage by infiltrating CD8+ T cells is Fas dependent.

Ag stimulation of CD8+ lymphocytes in vivo results in their migration to various tissues as well as the activation of a cytolytic program involving perforin, TNF-alpha, and Fas ligand. The liver is one of the main sites for infiltration by activated CD8+ T cells, and this is followed by the death of hepatocytes. The contribution of the various cytolytic components to this process is unclear. Hepatocyte damage by CD8+ T cells was studied using the MHC class I-restricted OVA-specific TCR transgenic mouse (OT-1) to examine the contribution of Fas to hepatocyte death. Activated CD8+ T cells from both OT-1 and Fas-deficient OT-1lpr mice migrated to the liver in similar numbers after OVA administration, but only in OT-1 mice was there evidence of significant hepatocyte damage histologically and by elevation of serum aspartate transaminase. These differences were not the result of inefficient induction of cytolytic activity in OT-1lpr liver T cells, since they were as cytolytic in vitro as OT-1 liver T cells. This was supported by findings of similar high levels of message for perforin, TNF-alpha, and Fas ligand in liver lymphocytes from both mice. These findings demonstrate that following Ag activation, infiltrating liver CD8+ T lymphocytes induce hepatocyte damage in a Fas-dependent manner.     

Type 1 and type 2 CD8+ effector T cell subpopulations promote long-term tumor immunity and protection to progressively growing tumor

Cytolytic CD8+ effector cells fall into two subpopulations based on cytokine secretion. Type 1 CD8+ T cells (Tc1) secrete IFN-gamma, whereas type 2 CD8+ T cells (Tc2) secrete IL-4, IL-5, and IL-10. Using an OVA-transfected B16 lung metastases model, we assessed the therapeutic effects of adoptively transferred OVA-specific Tc1 and Tc2 subpopulations in mice bearing established pulmonary malignancy. Effector cell-treated mice exhibiting high (5 x 105) tumor burdens experienced significant (p < 0.05) delays in mortality compared with those of untreated control mice, whereas high proportions (70-90%) of mice receiving therapy with low (1 x 105) tumor burdens survived indefinitely. Long-term tumor immunity was evident by resistance to lethal tumor rechallenge, heightened levels of systemic OVA Ag-specific CTL responses ex vivo, and detection of long-lived TCR transgene-positive donor cells accompanied by an elevation in the total numbers of CD8+ CD44high activated and/or memory T cells at sites of tumor growth. Long-lasting protection by Tc2 and Tc1 effector cells were dependent, in part, on both the level of tumor burden and effector cell-derived IL-4, IL-5, and IFN-gamma, respectively. We conclude that Tc1 and Tc2 effector cells provide immunity by different mechanisms that subsequently potentiate host-derived antitumor responses.     

Therapeutic effects of tumor-reactive type 1 and type 2 CD8+ T cell subpopulations in established pulmonary metastases.

Cytolytic CD8+ T cells fall into two subpopulations based on cytokine-secretion. Type 1 CD8+ cells (Tc1) characteristically secrete IFN-gamma, whereas type 2 CD8+ cells (Tc2) secrete IL-4 and IL-5. We assessed the relative therapeutic effects of adoptively transferred OVA-specific Tc1 and Tc2 CD8+ cells in mice bearing established OVA-transfected B16 melanoma lung metastases. Both Tc1 and Tc2 subpopulations mediated a reduction in lung tumor growth that subsequently prolonged survival times in mice with both early (day 7) and more advanced (day 14) levels of tumor development. CD8+ T cell populations recovered from spleens of tumor-bearing mice receiving Tc1 or Tc2 cells showed markedly enhanced tumor Ag-specific cytolytic and cytokine-releasing activities that correlated with delays in tumor cell growth and progression. Initially, both tumor-reactive Tc1 and Tc2 effector cells accumulated at the tumor site with nearly equal frequency. Tc1 cells persisted, whereas Tc2 cell numbers progressively diminished over time. Titration of Tc1 and Tc2 effector cells showed that protection was dose dependent with the former being 5-fold more effective. Tc2 cells achieved a comparable reduction in lung tumor cell growth at higher concentrations of cell transfer. Tc1 effectors from IFN-gamma-deficient mice were less therapeutically effective than wild-type mice, but there was no significant reduction in activity between corresponding Tc2 populations. We speculate that the effectiveness of Tc1 and Tc2 cells may depend on different mechanisms. These studies suggest a potential role for Tc1 and Tc2 CD8+ subpopulations in tumor regression and immunotherapy.     

Relative diabetogenic properties of islet-specific Tc1 and Tc2 cells in immunocompetent hosts

CD8(+) T cells are important effectors, as well as regulators, of organ-specific autoimmunity. Compared with Tc1-type CD8(+) cells, Tc2 cells have impaired anti-viral and anti-tumor effector functions, although no data are yet available on their pathogenic role in autoimmunity. Our aim was to explore the role of autoreactive Tc1 and Tc2 cells in autoimmune diabetes. We set up an adoptive transfer model in which the recipients were transgenic mice expressing influenza virus hemagglutinin (HA) specifically in their pancreatic ss islet cells (rat insulin promoter-HA mice) and islet-specific Tc1 and Tc2 cells were generated in vitro from HA-specific CD8(+) cells of TCR transgenic mice (CL4-TCR mice). One million Tc1 cells, differentiated in vitro in the presence of IL-12, transferred diabetes in 100% of nonirradiated adult rat insulin promoter-HA recipients; the 50% diabetogenic dose was 5 x 10(5). Highly polarized Tc2 cells generated in the presence of IL-4, IL-10, and anti-IFN-gamma mAb had a relatively low, but definite, diabetogenic potential. Thus, 5 x 10(6) Tc2 cells caused diabetes in 6 of 18 recipients, while the same dose of naive CD8(+) cells did not cause diabetes. Looking for the cause of the different diabetogenic potential of Tc1 and Tc2 cells, we found that Tc2 cells are at least as cytotoxic as Tc1 cells but their accumulation in the pancreas is slower, a possible consequence of differential chemokine receptor expression. The diabetogenicity of autoreactive Tc2 cells, most likely caused by their cytotoxic activity, precludes their therapeutic use as regulators of autoimmunity.     

IFN-gamma promotes Fas ligand- and perforin-mediated liver cell destruction by cytotoxic CD8 T cells

To study liver cell damage by CTL, CD8 T cells from P14 TCR transgenic (tg) mice specific for the gp33 epitope of lymphocytic choriomeningitis virus with either deficiency in IFN-gamma (P14.IFN-gamma(null)), functional Fas ligand (P14.gld), or perforin (P14.PKO) were transferred into H8 tg mice ubiquitously expressing gp33 Ag. Treatment of H8 recipient mice with agonistic anti-CD40 Abs induced vigorous expansion of the transferred P14 T cells and led to liver cell destruction determined by increase of glutamate dehydrogenase serum levels and induction of caspase-3 in hepatocytes. Liver injury was mediated by the Fas/Fas ligand (FasL) pathway and by perforin, because P14.gld and P14.PKO T cells failed to induce increased glutamate dehydrogenase levels despite strong in vivo proliferation. In addition, H8 tg mice lacking Fas were resistant to the pathogenic effect of P14 T cells. Besides FasL and perforin, IFN-gamma was also required for liver cell damage, because P14.IFN-gamma(null) T cells adoptively transferred into H8 mice failed to induce disease. Moreover, Fas expression on hepatocytes from H8 recipient mice was increased after transfer of wild-type compared with P14.IFN-gamma(null) T cells, and wild-type P14 T cells expressed higher levels of FasL than P14 T cells lacking IFN-gamma. Thus, our data suggest that IFN-gamma released by activated CD8 T cells upon Ag contact facilitates liver cell destruction.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit T cell-mediated cytotoxicity by inhibiting Fas ligand expression

We reported recently that the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) protect CD4+ T cells against Ag-induced apoptosis by down-regulating the expression of Fas ligand (FasL). Because the cytotoxic activity of CD8+ CTLs is mediated through two mechanisms, which involve the perforin/granzyme and the FasL/Fas pathways, in this study we investigated the effects of VIP/PACAP on the generation and activity of allogeneic CTLs, of CD8+ T1 and T2 effector cells and of alloreactive peritoneal exudate cytotoxic T cells (PEL) generated in vivo. VIP/PACAP did not affect perforin/granzyme-mediated cytotoxicity, perforin gene expression, or granzyme B enzymatic activity, but drastically inhibited FasL/Fas-mediated cytotoxicity against allogeneic or syngeneic Fas-bearing targets. VIP/PACAP inhibit CTL generation, but not the activity of competent CTLs. The inhibition is associated with a profound down-regulation of FasL expression, and these effects are mediated through both VPAC1 and VPAC2 receptors. VIP/PACAP inhibit the FasL/Fas-mediated cytotoxicity of T1 effectors and do not affect T2 cytotoxicity, which is entirely perforin/granzyme mediated. Similar effects were observed in vivo. Both the FasL/Fas-mediated cytotoxicity and FasL expression of cytotoxic allogeneic PELs generated in vivo in the presence of VIP or PACAP were significantly reduced. We conclude that, similar to their effect on CD4+ T cells, the two structurally related neuropeptides inhibit FasL expression in CD8+ cytotoxic T cells and the subsequent lysis of Fas-bearing target cells.     

Multifunctional CD4 cells expressing gamma interferon and perforin mediate protection against lethal influenza virus infection

CD4 effectors generated in vitro can promote survival against a highly pathogenic influenza virus via an antibody-independent mechanism involving class II-restricted, perforin-mediated cytotoxicity. However, it is not known whether CD4 cells activated during influenza virus infection can acquire cytolytic activity that contributes to protection against lethal challenge. CD4 cells isolated from the lungs of infected mice were able to confer protection against a lethal dose of H1N1 influenza virus A/Puerto Rico 8/34 (PR8). Infection of BALB/c mice with PR8 induced a multifunctional CD4 population with proliferative capacity and ability to secrete interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) in the draining lymph node (DLN) and gamma interferon (IFN-γ) and IL-10 in the lung. IFN-γ-deficient CD4 cells produced larger amounts of IL-17 and similar levels of TNF-α, IL-10, and IL-2 compared to wild-type (WT) CD4 cells. Both WT and IFN-γ(-/-) CD4 cells exhibit influenza virus-specific cytotoxicity; however, IFN-γ-deficient CD4 cells did not promote recovery after lethal infection as effectively as WT CD4 cells. PR8 infection induced a population of cytolytic CD4 effectors that resided in the lung but not the DLN. These cells expressed granzyme B (GrB) and required perforin to lyse peptide-pulsed targets. Lethally infected mice given influenza virus-specific CD4 cells deficient in perforin showed greater weight loss and a slower time to recovery than mice given WT influenza virus-specific CD4 cells. Taken together, these data strengthen the concept that CD4 T cell effectors are broadly multifunctional with direct roles in promoting protection against lethal influenza virus infection.     

CD4 T cell-mediated protection from lethal influenza: perforin and antibody-mediated mechanisms give a one-two punch

The mechanisms whereby CD4 T cells contribute to the protective response against lethal influenza infection remain poorly characterized. To define the role of CD4 cells in protection against a highly pathogenic strain of influenza, virus-specific TCR transgenic CD4 effectors were generated in vitro and transferred into mice given lethal influenza infection. Primed CD4 effectors conferred protection against lethal infection over a broad range of viral dose. The protection mediated by CD4 effectors did not require IFN-gamma or host T cells, but did result in increased anti-influenza Ab titers compared with untreated controls. Further studies indicated that CD4-mediated protection at high doses of influenza required B cells, and that passive transfer of anti-influenza immune serum was therapeutic in B cell-deficient mice, but only when CD4 effectors were present. Primed CD4 cells also acquired perforin (Pfn)-mediated cytolytic activity during effector generation, suggesting a second mechanism used by CD4 cells to confer protection. Pfn-deficient CD4 effectors were less able to promote survival in intact BALB/c mice and were unable to provide protection in B cell-deficient mice, indicating that Ab-independent protection by CD4 effectors requires Pfn. Therefore, CD4 effectors mediate protection to lethal influenza through at least two mechanisms: Pfn-mediated cytotoxicity early in the response promoted survival independently of Ab production, whereas CD4-driven B cell responses resulted in high titer Abs that neutralized remaining virus.     

A Coreceptor-Independent Transgenic Human TCR Mediates Anti-Tumor and Anti-Self Immunity in Mice

Recent advancements in T cell immunotherapy suggest that T cells engineered with high-affinity TCR can offer better tumor regression. However, whether a high-affinity TCR alone is sufficient to control tumor growth, or the T cell subset bearing the TCR is also important remains unclear. Using the human tyrosinase epitope-reactive, CD8-independent, high-affinity TCR isolated from MHC class I-restricted CD4(+) T cells obtained from tumor-infiltrating lymphocytes (TIL) of a metastatic melanoma patient, we developed a novel TCR transgenic mouse with a C57BL/6 background. This HLA-A2-restricted TCR was positively selected on both CD4(+) and CD8(+) single-positive cells. However, when the TCR transgenic mouse was developed with a HLA-A2 background, the transgenic TCR was primarily expressed by CD3(+)CD4(-)CD8(-) double-negative T cells. TIL 1383I TCR transgenic CD4(+), CD8(+), and CD4(-)CD8(-) T cells were functional and retained the ability to control tumor growth without the need for vaccination or cytokine support in vivo. Furthermore, the HLA-A2(+)/human tyrosinase TCR double-transgenic mice developed spontaneous hair depigmentation and had visual defects that progressed with age. Our data show that the expression of the high-affinity TIL 1383I TCR alone in CD3(+) T cells is sufficient to control the growth of murine and human melanoma, and the presence or absence of CD4 and CD8 coreceptors had little effect on its functional capacity.     

Perforin and Fas cytolytic pathways coordinately shape the selection and diversity of CD8+-T-cell escape variants of influenza virus

Antigenic variation is a viral strategy exploited to promote survival in the face of the host immune response and represents a major challenge for efficient vaccine development. Influenza viruses are pathogens with high transmissibility and mutation rates, enabling viral escape from immunity induced by prior infection or vaccination. Intense selection from neutralizing antibody drives antigenic changes in the surface glycoproteins, resulting in emergence of new strains able to reinfect hosts immune to previously circulating viruses. CD8+ cytotoxic T cells (CTLs) also provide protective immunity from influenza virus infection and may contribute to the antigenic evolution of influenza viruses. Utilizing mice transgenic for an influenza virus NP366-374 peptide-specific T-cell receptor, we demonstrated that the respiratory tract is a suitable site for generation of escape variants of influenza virus selected by CTL in vivo. In this report the contributions of the perforin and Fas pathways utilized by influenza virus-specific CTLs in viral clearance and selection of CTL escape variants have been evaluated. While transgenic CTLs deficient in either perforin- or Fas-mediated pathways are efficient in initial pulmonary viral control, variant virus emergence was observed in all the mice studied, although the spectrum of viral CTL escape variants selected varied profoundly. Thus, a less-restricted repertoire of escape variants was observed in mice with an intact perforin cytotoxic pathway compared with a limited variant diversity in perforin pathway-deficient mice, although maximal variant diversity was observed in mice having both Fas and perforin pathways intact. We conclude that selection of viral CTL escape variants reflects coordinate action between the tightly controlled perforin/granzyme pathway and the more promiscuous Fas/FasL pathway.