Inhibition of the death receptor pathway by cFLIP confers partial engraftment of MHC class I-deficient stem cells and reduces tumor clearance in perforin-deficient mice

NK cells mediate acute rejection of MHC class I-deficient bone marrow cell (BMC) grafts. However, the exact cytotoxic mechanisms of NK cells during acute BMC graft rejection are not well defined. Although the granule exocytosis pathway plays a major role in NK cell-mediated rejection, alternative perforin-independent mechanisms also exist. By analyzing the anti-apoptotic effects of cellular Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein (cFLIP) overexpression, we investigated the possible role of death receptor-induced apoptosis in NK cell-mediated cytotoxicity. In the absence of perforin, we found that cFLIP overexpression reduces lysis of tumor cells by NK cells in vitro and in vivo. In addition, perforin-deficient NK cells were impaired in their ability to acutely reject cFLIP-overexpressing TAP-1 knockout stem cells. These results emphasize the importance of NK cell death receptor-mediated killing during BMC grafts in the absence of perforin.     

Ly49I NK cell receptor transgene inhibition of rejection of H2b mouse bone marrow transplants

The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6+ NK cells prevents rejection of allogeneic or parental-strain H2d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49IB6). Ly49IB6 is ITIM+ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2q mice reject H2b but not H2d BMC allografts, and the rejection of H2b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB. Ly49IB6 hosts failed to reject H2d or H2b BMC, but did reject class I-deficient TAP-1-/- BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2b BMC by FVB.Ly49IB6 mice. FVB.Ly49IB6+/- mice were crossed and back-crossed with 129 mice-H2b, 5E6-, poor responders to H2d BMC grafts. While transgene-negative H2b/q F1 or first-generation back-crossed mice rejected H2b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2b MHC class I molecules. Moreover, Ly49IB6 has no positive influence on the rejection of H2d allografts.     

Perforin-dependent cytolytic responses in beta2-microglobulin-deficient mice.

The ability of beta2-microglobulin-deficient mice (B6.beta2micro(o)) mice to reject syngeneic and major histocompatability (MHC) class I-deficient tumor grafts was examined with a view to determining residual cytotoxic activities that exist in these mice. In particular, the cytotoxic activities of NK cells and CD8(+) cytotoxic T lymphocytes (CTL) reactive against self-MHC class I were assessed using a variety of gene-targeted mice. The creation of mice doubly deficient for perforin and beta2micro (B6.P(o).beta2micro(o)) enabled the determination that perforin was responsible for the cytotoxic activity of NK cells and CD8(+) CTL reactive against self-MHC class I. Dependence on perforin function was demonstrated for the cytotoxicity of these effectors in vitro and for the ability of these effectors to reject a variety of tumors in vivo.     

Functional defects of NK cells treated with chloroquine mimic the lytic defects observed in perforin-deficient mice

NK cells are the primary effectors mediating acute rejection of incompatible bone marrow cell grafts. To reduce rejection, we evaluated the ability of chloroquine (CHQ) to prevent perforin-dependent NK cell activity. Perforin is a key cytotoxic component released from the lytic granules of activated NK cells. Generation of functional perforin requires an acidic protease activity that occurs in the secretory, lytic lysosomes. Our hypothesis was that CHQ, a lysosomotropic reagent, would raise the pH of the acidic compartment in which perforin is processed and thereby block perforin maturation and cytotoxicity. We have measured NK cytotoxicity in vivo by clearance of YAC-1 tumor cells from the lungs and by rejection of incompatible bone marrow transplants and in vitro by cytolysis of YAC-1 and Jurkat cells. The engraftment of bone marrow cells was monitored by recolonization of the spleen with hemopoietic cells from transplants of MHC class I-deficient bone marrow cells into lethally irradiated recipient mice. Transplant rejection was compared in two inbred strains of mice: 129, which apparently use perforin-dependent cytotoxicity, and C57BL/6, in which rejection can be perforin-independent. CHQ treatment reduced NK cell activity in 129 mice in which perforin is important for mediating rejection. CHQ affected the fraction of NK cell cytolysis that was Fas independent. In addition, we found that CHQ prevents perforin processing by LAK cells in vitro. These data indicate that CHQ may impair rejection of incompatible bone marrow transplants and other functions mediated by NK and cytotoxic T cells.     

A central role for death receptor-mediated apoptosis in the rejection of tumors by NK cells

NK cells provide a line of defense against tumors and virus-infected cells that have lost the expression of one or more MHC class I isoforms. Here, we investigate whether inhibitors of apoptosis can block the rejection of tumors mediated by NK cells, by introducing the long form of Fas-associated death domain-like IL-1beta-converting enzyme-associated inhibitory protein (FLIP(L)) and poxvirus cytokine response modifier A (CrmA) into the MHC class I-deficient T lymphoma cell line RMA-S. RMA-S cells do not normally express Fas in vitro, and it was previously postulated that the rejection of these tumors by NK cells is strictly perforin dependent. We show that perforin-deficient NK cells directly mediate Fas up-regulation on RMA-S cells and thereafter kill the cells in a Fas-dependent manner, and that RMA-S FLIP(L) and RMA-S CrmA are protected from such killing. When injected in immunocompetent recipients, RMA-S cells up-regulate Fas, rendering in vivo-passed mock-transduced cells sensitive to Fas-mediated apoptosis. Moreover, RMA-S FLIP(L) and RMA-S CrmA cells establish aggressive tumors, in contrast to RMA-S mock cells that are rejected. These results demonstrate that FLIP(L) and CrmA function as tumor progression factors by protecting MHC class I-deficient tumors from rejection mediated by NK cells. Moreover, our data indicate that death receptor-mediated apoptosis has a more prominent role in the clearance of NK-sensitive tumors than previously suggested.     

A role for IFN-gamma in primary and secondary immunity generated by NK cell-sensitive tumor-expressing CD80 in vivo

We have investigated the primary and secondary immunity generated in vivo by a MHC class I-deficient tumor cell line that expressed CD80 (B7-1). CD80 expression enhanced primary NK cell-mediated tumor rejection in vivo and T cell immunity against secondary tumor challenge. CD80 expression enhanced primary NK cell-mediated tumor rejection, and both NK cell perforin and IFN-gamma activity were critical for the rejection of MHC class I-deficient RMA-S-CD80 tumor cells. This primary rejection process stimulated the subsequent development of specific CTL and Th1 responses against Ags expressed by the MHC class I-deficient RMA-S tumor cells. The development of effective secondary T cell immunity could be elicited by irradiated RMA-S-CD80 tumor cells and was dependent upon NK cells and IFN-gamma in the priming response. Our findings demonstrate a key role for IFN-gamma in innate and adaptive immunity triggered by CD80 expression on tumor cells.     

Cutting edge: tumor rejection mediated by NKG2D receptor-ligand interaction is dependent upon perforin

We have investigated the primary immunity generated in vivo by MHC class I-deficient and -competent tumor cell lines that expressed the NKG2D ligand retinoic acid early inducible-1 (Rae-1) beta. Rae-1beta expression on class I-deficient RMA-S lymphoma cells enhanced primary NK cell-mediated tumor rejection in vivo, whereas RMA-Rae-1beta tumor cells were rejected by a combination of NK cells and CD8(+) T cells. Rae-1beta expression stimulated NK cell cytotoxicity and IFN-gamma secretion in vitro, but not proliferation. Surprisingly, only NK cell perforin-mediated cytotoxicity, but not production of IFN-gamma, was critical for the rejection of Rae-1beta-expressing tumor cells in vivo. This distinct requirement for perforin activity contrasts with the NK cell-mediated rejection of MHC class I-deficient RMA-S tumor cells expressing other activating ligands such as CD70 and CD80. Thus, these results indicated that NKG2D acted as a natural cytotoxicity receptor to stimulate perforin-mediated elimination of ligand-expressing tumor cells.     

Cutting edge: perforin down-regulates CD4 and CD8 T cell-mediated immune responses to a transplanted organ

Perforin mediates target cell apoptosis by CTLs and NK cells. Although perforin expression correlates strongly with acute allograft rejection, perforin-deficient mice reject allografts with the same kinetics as wild-type recipients. In this study, we tested the hypothesis that while perforin is dispensable for acute rejection, it is essential for down-regulating the alloimmune response by inducing the apoptosis of host immune cells. Using a skin transplantation model, we found that perforin-deficient mice are resistant to the induction of allograft acceptance by agents that block T cell costimulation. Failure to induce allograft acceptance in these mice was observed irrespective of whether the alloimmune response was CD4 or CD8 T cell-mediated and could be attributed to defective apoptosis of activated CD4 and CD8 T cells. In contrast, perforin did not influence T cell proliferation. Therefore, perforin is an essential immunoregulatory molecule that may be required for the induction of transplantation tolerance.     

MHC Class I Expression by Donor Hematopoietic Stem Cells Is Required to Prevent NK Cell Attack in Allogeneic,but Not Syngeneic Recipient Mice

NK cells resist engraftment of syngeneic and allogeneic bone marrow (BM) cells lacking major histocompatibility (MHC) class I molecules, suggesting a critical role for donor MHC class I molecules in preventing NK cell attack against donor hematopoietic stem and progenitor cells (HSPCs), and their derivatives. However, using high-resolution in vivo imaging, we demonstrated here that syngeneic MHC class I knockout (KO) donor HSPCs persist with the same survival frequencies as wild-type donor HSPCs. In contrast, syngeneic MHC class I KO differentiated hematopoietic cells and allogeneic MHC class I KO HSPCs were rejected in a manner that was significantly inhibited by NK cell depletion. In vivo time-lapse imaging demonstrated that mice receiving allogeneic MHC class I KO HSPCs showed a significant increase in NK cell motility and proliferation as well as frequencies of NK cell contact with and killing of HSPCs as compared to mice receiving wild-type HSPCs. The data indicate that donor MHC class I molecules are required to prevent NK cell-mediated rejection of syngeneic differentiated cells and allogeneic HSPCs, but not of syngeneic HSPCs.     

NK cells promote islet allograft tolerance via a perforin-dependent mechanism

Although major histocompatibility complex (MHC) class II-restricted CD4 T cells are well appreciated for their contribution to peripheral tolerance to tissue allografts, little is known regarding MHC class I-dependent reactivity in this process. Here we show a crucial role for host MHC class I-dependent NK cell reactivity for allograft tolerance in mice induced through either costimulation blockade using CD154-specific antibody therapy or by targeting LFA-1 (also known as CD11a). Tolerance induction absolutely required host expression of MHC class I, but was independent of CD8 T cell-dependent immunity. Rather, tolerance required innate immunity involving NK1.1(+) cells, but was independent of CD1d-restricted NKT cells. Therefore, NK cells seem to be generally required for induction of tolerance to islet allografts. Additional studies indicate that CD154-specific antibody-induced allograft tolerance is perforin dependent. Notably, NK cells that are perforin competent are sufficient to restore allograft tolerance in perforin-deficient recipients. Together, these results show an obligatory role for NK cells, through perforin, for induction of tolerance to islet allografts.     

A nonclassical MHC class I molecule restricts CTL-mediated rejection of a syngeneic melanoma tumor

Although CTL and polymorphic, classical MHC class I molecules have well defined roles in the immune response against tumors, little is currently known regarding the participation of nonpolymorphic, nonclassical MHC class I in antitumor immunity. Using an MHC class I-deficient melanoma as a model tumor, we demonstrate that Q9, a murine MHC class Ib molecule from the Qa-2 family, expressed on the surface of tumor cells, protects syngeneic hosts from melanoma outgrowth. Q9-mediated protective immunity is lost or greatly diminished in mice deficient in CTL, including beta(2)-microglobulin knockout (KO), CD8 KO, and SCID mice. In contrast, the Q9 antitumor effects are not detectably suppressed in CD4 KO mice with decreased Th cell activity. Killing by antitumor CTL in vitro is Q9 specific and can be blocked by anti-Q9 and anti-CD8 Abs. The adaptive Q9-restricted CTL response leads to immunological memory, because mice that resist the initial tumor challenge reject subsequent challenges with less immunogenic tumor variants and show expansion of CD8(+) T cell populations with an activated/memory CD44(high) phenotype. Collectively, these studies demonstrate that a MHC class Ib molecule can serve as a restriction element for antitumor CTL and mediate protective immune responses in a syngeneic setting.     

Perforin is a major contributor to NK cell control of tumor metastasis.

We provide the first demonstration, using experimental and spontaneous models of metastasis in C57BL/6 (B6) (RM-1 prostate carcinoma) and BALB/c (DA3 mammary carcinoma) mice, that tumor metastasis is primarily controlled by perforin-dependent cytotoxicity mediated by NK1.1+ cells. MHC class Ilow RM-1 and DA3 tumor cells were sensitive in vitro to Fas-mediated lysis or spleen NK cells in a perforin-dependent fashion. Perforin-deficient NK cells did not lyse these tumors, and perforin-deficient mice were 10-100-fold less proficient than wild-type mice in rejecting the metastasis of tumor cells to the lung. Fas ligand mutant gld mice displayed uncompromised protection against tumor metastasis. Depletion of NK subsets resulted in greater numbers of metastases than observed in perforin-deficient mice, suggesting that perforin-independent effector functions of NK cells may also contribute to protection from tumor metastasis.     

Perforin-mediated effector function within the central nervous system requires IFN-gamma-mediated MHC up-regulation

CD8(+) T cells infiltrating the CNS control infection by the neurotropic JHM strain of mouse hepatitis virus. Differential susceptibility of infected cell types to clearance by perforin or IFN-gamma uncovered distinct, nonredundant roles for these antiviral mechanisms. To separately evaluate each effector function specifically in the context of CD8(+) T cells, pathogenesis was analyzed in mice deficient in both perforin and IFN-gamma (PKO/GKO) or selectively reconstituted for each function by transfer of CD8(+) T cells. Untreated PKO/GKO mice were unable to control the infection and died of lethal encephalomyelitis within 16 days, despite substantially higher CD8(+) T cell accumulation in the CNS compared with controls. Uncontrolled infection was associated with limited MHC class I up-regulation and an absence of class II expression on microglia, coinciding with decreased CD4(+) T cells in CNS infiltrates. CD8(+) T cells from perforin-deficient and wild-type donors reduced virus replication in PKO/GKO recipients. By contrast, IFN-gamma-deficient donor CD8(+) T cells did not affect virus replication. The inability of perforin-mediated mechanisms to control virus in the absence of IFN-gamma coincided with reduced class I expression. These data not only confirm direct antiviral activity of IFN-gamma within the CNS but also demonstrate IFN-gamma-dependent MHC surface expression to guarantee local T cell effector function in tissues inherently low in MHC expression. The data further imply that IFN-gamma plays a crucial role in pathogenesis by regulating the balance between virus replication in oligodendrocytes, CD8(+) T cell effector function, and demyelination.     

Rejection of mouse cardiac allografts by costimulation in trans

The activation of T cells by B7 costimulation in trans has been demonstrated in vitro, but the in vivo relevance is unknown. To study costimulation in trans of CD4(+) T cells in vivo, we performed cardiac transplants from B7-1/B7-2-deficient mice to recipients that do not express MHC class II molecules on peripheral APCs, but do have functional CD4(+) T cells (II(-)/4(+) mice). This model restricts the B7-dependent activation of CD4(+) T cells to costimulation in trans and excludes any contribution from indirect Ag presentation. We find that II(-)/4(+) recipients reject B7-deficient grafts as rapidly as wild-type grafts, suggesting that costimulation in trans can mediate rejection as potently as costimulation in cis. Treatment of II(-)/4(+) recipients of B7-deficient grafts with depleting Abs to CD4 or CD8 demonstrates that indirect Ag presentation to CD8(+) cells does not significantly contribute to rejection. This is the first demonstration that costimulation in trans can mediate an immune response in vivo and has important therapeutic implications.     

NK cells play a critical role in the regulation of class I-deficient hemopoietic stem cell engraftment: evidence for NK tolerance correlates with receptor editing

The role that NK cells play in the rejection of hemopoietic stem cell (HSC) and tolerance induction has remained controversial. In this study, we examined whether NK cells play a direct role in the rejection of HSC. Purified HSC from MHC class II-deficient mice engrafted readily in congenic mice, while HSC from class I-deficient donors (beta(2)-microglobulin(-/-) (beta(2)m(-/-))) failed to engraft. Recipient mice lacking CD8(+), CD4(+), or T cells also rejected HSC from class I-deficient donors, pointing directly to NK cells as the effector in rejection of HSC. Recipients, deficient in or depleted of NK cells, engrafted readily with beta(2)m(-/-) HSC. Expression of the activating Ly-49D and inhibitory Ly-49G2 receptors on recipient NK cells was significantly decreased in these beta(2)m(-/-)-->B6 chimeras, and the proportion of donor NK cells expressing Ly-49D was also significantly decreased. Notably, beta(2)m(-/-) chimeras accepted beta(2)m(-/-) HSC in second transplants, demonstrating that NK cells in the chimeras had been tolerized to beta(2)m(-/-). Taken together, our data demonstrate that NK cells play a direct role in the regulation of HSC engraftment, and down-regulation and/or deletion of specific NK subsets in mixed chimeras can contribute to the induction of NK cell tolerance in vivo. Moreover, our data show that bone marrow-derived elements significantly contribute to NK cell development and tolerance.     

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.     

Oral administration of high molecular mass poly-gamma-glutamate induces NK cell-mediated antitumor immunity

We analyzed the in vivo tumor regression activity of high molecular mass poly-gamma-glutamate (gamma-PGA) from Bacillus subtilis sups. chungkookjang. C57BL/6 mice were orally administered 10-, 100-, or 2000-kDa gamma-PGA or beta-glucan (positive control), and antitumor immunity was examined. Our results revealed higher levels of NK cell-mediated cytotoxicity and IFN-gamma secretion in mice treated with higher molecular mass gamma-PGA (2000 kDa) vs those treated with lower molecular mass gamma-PGA (10 or 100 kDa) or beta-glucan. We then examined the effect of oral administration of 10- or 2000-kDa gamma-PGA on protection against B16 tumor challenge in C57BL/6 mice. Mice receiving high molecular mass gamma-PGA (2000 kDa) showed significantly smaller tumor sizes following challenge with the MHC class I-down-regulated tumor cell lines, B16 and TC-1 P3 (A15), but not with TC-1 cells, which have normal MHC class I expression. Lastly, we found that gamma-PGA-induced antitumor effect was decreased by in vivo depletion of NK cells using mAb PK136 or anti-asialo GM1 Ab, and that was completely blocked in NK cell-deficient B6 beige mice or IFN-gamma knockout mice. Taken together, we demonstrated that oral administration of high molecular mass gamma-PGA (2000 kDa) generated significant NK cell-mediated antitumor activity in mice bearing MHC class I-deficient tumors.     

CD8+ T cells circumvent immune privilege in the eye and mediate intraocular tumor rejection by a TNF-alpha-dependent mechanism

Although intraocular tumors reside in an immune-privileged environment, T cells can circumvent immune privilege and mediate tumor rejection without inducing damage to normal ocular tissue. In this study, we used a well-characterized tumor, Ad5E1 (adenovirus type 5 early region 1), to analyze the role of CD8+ T cells in the pristine rejection of intraocular tumors. It has been previously documented that Ad5E1 tumor rejection can occur in the absence of CD8+ T cells. However, here we find that CD8+ T cells infiltrated intraocular Ad5E1 tumors in C57BL/6 mice. Surprisingly, CD8+ T cells from tumor-rejector mice could mediate intraocular tumor rejection following adoptive transfer to SCID mice. In determining the mechanisms behind CD8+ T cell-mediated tumor rejection, we discovered that antitumor CTL activity was neither observed nor necessary for rejection of the intraocular tumors. CD8+ T cells from rejector mice did not produce IFN-gamma in response to Ad5E1 tumor Ags or use FasL to mediate intraocular tumor rejection. Also, CD8+ T cells did not use perforin or TRAIL, as CD8+ T cells from perforin knockout (KO) and TRAIL KO mice conferred protection to SCID recipient mice following adoptive transfer. We discovered that CD8+ T cells used TNF-alpha to mediate tumor rejection, because Ad5E1 tumor cells were highly sensitive to TNF-alpha-induced apoptosis and CD8+ T cells from TNF-alpha KO mice did not protect SCID mice from progressive Ad5E1 tumor growth. The results indicate that CD8+ T cells circumvent immune privilege and mediate intraocular tumor rejection by a TNF-alpha-dependent manner while leaving the eye intact and vision preserved.     

Adaptive immunity and enhanced CD8+ T cell response to Listeria monocytogenes in the absence of perforin and IFN-gamma

Single Ag-specific CD8+ T cells from IFN-gamma-deficient (GKO) or perforin-deficient (PKO) mice provide substantial immunity against murine infection with Listeria monocytogenes. To address the potential for redundancy between perforin and IFN-gamma as CD8+ T cell effector mechanisms, we generated perforin/IFN-gamma (PKO/GKO) double-deficient mice. PKO/GKO-derived CD8+ T cells specific for the immunodominant listeriolysin O (LLO91-99) epitope provide immunity to LM infection similar to that provided by Ag-matched wild-type (WT) CD8+ T cells in the liver but reduced in the spleen. Strikingly, polyclonal CD8+ T cells from immunized PKO/GKO mice were approximately 100-fold more potent in reducing bacterial numbers than the same number of polyclonal CD8+ T cells from immunized WT mice. This result is probably quantitative, because the frequency of the CD8+ T cell response against the immunodominant LLO91-99 epitope is >4.5-fold higher in PKO/GKO mice than WT mice at 7 days after identical immunizations. Moreover, PKO/GKO mice can be immunized by a single infection with attenuated Listeria to resist >80,000-fold higher challenges with virulent organisms than naive PKO/GKO mice. These data demonstrate that neither perforin nor IFN-gamma is required for the development or expression of adaptive immunity to LM. In addition, the results suggest the potential for perforin and IFN-gamma to regulate the magnitude of the CD8+ T cell response to infection.