Synergistic Reversal of Intrahepatic HCV-Specific CD8 T Cell Exhaustion by Combined PD-1/CTLA-4 Blockade

Viral persistence is associated with hierarchical antiviral CD8 T cell exhaustion with increased programmed death-1 (PD-1) expression. In HCV persistence, HCV-specific CD8 T cells from the liver (the site of viral replication) display increased PD-1 expression and a profound functional impairment that is not reversed by PD-1 blockade alone. Here, we report that the inhibitory receptor cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is preferentially upregulated in PD-1⁺ T cells from the liver but not blood of chronically HCV-infected patients. PD-1/CTLA-4 co-expression in intrahepatic T cells was associated with a profound HCV-specific effector dysfunction that was synergistically reversed by combined PD-1/CTLA-4 blockade in vitro, but not by blocking PD-1 or CTLA-4 alone. A similar effect was observed in circulating HCV-specific CD8 T cells with increased PD-1/CTLA-4 co-expression during acute hepatitis C. The functional response to combined blockade was directly associated with CTLA-4 expression, lost with CD28-depletion and CD4-independent (including CD4⁺FoxP3⁺ Tregs). We conclude that PD-1 and CTLA-4 pathways both contribute to virus-specific T cell exhaustion at the site of viral replication by a redundant mechanism that requires combined PD-1/CTLA-4 blockade to reverse. These findings provide new insights into the mechanisms of virus-specific T cell dysfunction, and suggest that the synergistic effect by combined inhibitory receptor blockade might have a therapeutic application against chronic viral infection in vivo, provided that it does not induce autoimmunity.     

CD8+ T Cell Priming by Dendritic Cell Vaccines Requires Antigen Transfer to Endogenous Antigen Presenting Cells

Background

Immunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs) that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression.

Methodology/Principal Findings

We examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells.

Conclusions/Significance

This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs.     

Bystander Activation and Anti-Tumor Effects of CD8+ T Cells Following Interleukin-2 Based Immunotherapy Is Independent of CD4+ T Cell Help

We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent “bystander-activated” (CD8⁺CD44^high) T cells displaying a CD25⁻NKG2D⁺ phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4+ T cell help for antigen-specific CD8+ T cell expansion, little is known regarding the role of CD4+ T cells in antigen-nonspecific bystander-memory CD8+ T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8+ T cells upregulated PD-1 in the absence of CD4+ T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8+ T cells. Interestingly, compared to CD8+ T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8+ response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8+ T cell expansion, CD4+ T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8+ T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion.     

CD40 Activation Rescues Antiviral CD8+ T Cells from PD-1-Mediated Exhaustion

The intrahepatic immune environment is normally biased towards tolerance. Nonetheless, effective antiviral immune responses can be induced against hepatotropic pathogens. To examine the immunological basis of this paradox we studied the ability of hepatocellularly expressed hepatitis B virus (HBV) to activate immunologically naïve HBV-specific CD8⁺ T cell receptor (TCR) transgenic T cells after adoptive transfer to HBV transgenic mice. Intrahepatic priming triggered vigorous in situ T cell proliferation but failed to induce interferon gamma production or cytolytic effector function. In contrast, the same T cells differentiated into cytolytic effector T cells in HBV transgenic mice if Programmed Death 1 (PD-1) expression was genetically ablated, suggesting that intrahepatic antigen presentation per se triggers negative regulatory signals that prevent the functional differentiation of naïve CD8⁺ T cells. Surprisingly, coadministration of an agonistic anti-CD40 antibody (αCD40) inhibited PD-1 induction and restored T cell effector function, thereby inhibiting viral gene expression and causing a necroinflammatory liver disease. Importantly, the depletion of myeloid dendritic cells (mDCs) strongly diminished the αCD40 mediated functional differentiation of HBV-specific CD8⁺ T cells, suggesting that activation of mDCs was responsible for the functional differentiation of HBV-specific CD8⁺ T cells in αCD40 treated animals. These results demonstrate that antigen-specific, PD-1-mediated CD8⁺ T cell exhaustion can be rescued by CD40-mediated mDC-activation.     

CD4+ and CD8+ T Cell Activation Are Associated with HIV DNA in Resting CD4+ T Cells

The association between the host immune environment and the size of the HIV reservoir during effective antiretroviral therapy is not clear. Progress has also been limited by the lack of a well-accepted assay for quantifying HIV during therapy. We examined the association between multiple measurements of HIV and T cell activation (as defined by markers including CD38, HLA-DR, CCR5 and PD-1) in 30 antiretroviral-treated HIV-infected adults. We found a consistent association between the frequency of CD4⁺ and CD8⁺ T cells expressing HLA-DR and the frequency of resting CD4+ T cells containing HIV DNA. This study highlights the need to further examine this relationship and to better characterize the biology of markers commonly used in HIV studies. These results may also have implications for reactivation strategies.     

Antigen Presenting B Cells Facilitate CD4 T Cell Cooperation Resulting in Enhanced Generation of Effector and Memory CD4 T Cells

We show that the in vivo generation of cytokine-producing CD4 T cells specific for a given major histocompatibility class-II (MHCII)-binding peptide of hen egg lysozyme (HEL) is facilitated when mice are immunized with splenic antigen presenting cells (APC) pulsed with this HEL peptide and another peptide that binds a different MHCII molecule. This enhanced generation of peptide-specific effector CD4 T cells requires that the same splenic APC be pulsed with both peptides. Pulsed B cells, but not pulsed dendritic cells (DCs), can mediate CD4 T cell cooperation, which can be blocked by disrupting OX40-OX40L (CD134-CD252) interactions. In addition, the generation of HEL peptide-specific CD4 T cell memory is greater when mice are primed with B cells pulsed with the two peptides than with B cells pulsed with the HEL- peptide alone. Based on our findings, we suggest CD4 T cell cooperation is important for vaccine design, underlies the phenomenon of “epitope-spreading” seen in autoimmunity, and that the efficacy of B cell-depletion in the treatment of human cell-mediated autoimmune disease is due to the abrogation of the interactions between autoimmune CD4 T cells that facilitates their activation.     

PD-1 and Tim-3 Pathways Regulate CD8+ T Cells Function in Atherosclerosis

T cell-mediated immunity plays a significant role in the development of atherosclerosis (AS). There is increasing evidence that CD8⁺ T cells are also involved in AS but their exact roles remain unclear. The inhibitory receptors programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain 3 (Tim-3) are well known inhibitory molecules that play a crucial role in regulating CD8⁺ T cell activation or tolerance. Here, we demonstrate that the co-expression of PD-1 and Tim-3 on CD8⁺ T cells is up-regulated in AS patients. PD-1⁺ Tim-3⁺ CD8⁺ T cells are enriched for within the central T (T_CM) cell subset, with high proliferative activity and CD127 expression. Co-expression of PD-1 and Tim-3 on CD8⁺ T cells is associated with increased anti-atherogenic cytokine production as well as decreased pro-atherogenic cytokine production. Blockade of PD-1 and Tim-3 results in a decrease of anti-atherogenic cytokine production by PD-1⁺ Tim-3⁺ CD8⁺ T cells and in an augmentation of TNF-α and IFN-γ production. These findings highlight the important role of the PD-1 and Tim-3 pathways in regulating CD8⁺ T cells function in human AS.     

IgE-Mediated Enhancement of CD4+ T Cell Responses in Mice Requires Antigen Presentation by CD11c+ Cells and Not by B Cells

IgE antibodies, administered to mice together with their specific antigen, enhance antibody and CD4⁺ T cell responses to this antigen. The effect is dependent on the low affinity receptor for IgE, CD23, and the receptor must be expressed on B cells. In vitro, IgE-antigen complexes are endocytosed via CD23 on B cells, which subsequently present the antigen to CD4⁺ T cells. This mechanism has been suggested to explain also IgE-mediated enhancement of immune responses in vivo. We recently found that CD23⁺ B cells capture IgE-antigen complexes in peripheral blood and rapidly transport them to B cell follicles in the spleen. This provides an alternative explanation for the requirement for CD23⁺ B cells. The aim of the present study was to determine whether B-cell mediated antigen presentation of IgE-antigen complexes explains the enhancing effect of IgE on immune responses in vivo. The ability of spleen cells, taken from mice 1–4 h after immunization with IgE-antigen, to present antigen to specific CD4⁺ T cells was analyzed. Antigen presentation was intact when spleens were depleted of CD19⁺ cells (i.e., primarily B cells) but was severely impaired after depletion of CD11c⁺ cells (i.e., primarily dendritic cells). In agreement with this, the ability of IgE to enhance proliferation of CD4⁺ T cells was abolished in CD11c-DTR mice conditionally depleted of CD11c⁺ cells. Finally, the lack of IgE-mediated enhancemen of CD4⁺ T cell responses in CD23^-/- mice could be rescued by transfer of MHC-II-compatible as well as by MHC-II-incompatible CD23⁺ B cells. These findings argue against the idea that IgE-mediated enhancement of specific CD4⁺ T cell responses in vivo is caused by increased antigen presentation by B cells. A model where CD23⁺ B cells act as antigen transporting cells, delivering antigen to CD11c⁺ cells for presentation to T cells is consistent with available experimental data.     

Cellular Size as a Means of Tracking mTOR Activity and Cell Fate of CD4+ T Cells upon Antigen Recognition

mTOR is a central integrator of metabolic and immunological stimuli, dictating immune cell activation, proliferation and differentiation. In this study, we demonstrate that within a clonal population of activated T cells, there exist both mTOR^hi and mTOR^lo cells exhibiting highly divergent metabolic and immunologic functions. By taking advantage of the role of mTOR activation in controlling cellular size, we demonstrate that upon antigen recognition, mTOR^hi CD4+ T cells are destined to become highly glycolytic effector cells. Conversely, mTOR^lo T cells preferentially develop into long-lived cells that express high levels of Bcl-2, CD25, and CD62L. Furthermore, mTOR^lo T cells have a greater propensity to differentiate into suppressive Foxp3+ T regulatory cells, and this paradigm was also observed in human CD4+ T cells. Overall, these studies provide the opportunity to track the development of effector and memory T cells from naïve precursors, as well as facilitate the interrogation of immunologic and metabolic programs that inform these fates.     

Viral Sequestration of Antigen Subverts Cross Presentation to CD8+ T Cells

Virus-specific CD8⁺ T cells (T_CD8+) are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC). Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector T_CD8+. Direct presentation of vaccinia virus (VACV) antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated T_CD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the T_CD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation must also be taken into account during the rational design of antiviral vaccines.     

A Novel HLA-B18 Restricted CD8+ T Cell Epitope Is Efficiently Cross-Presented by Dendritic Cells from Soluble Tumor Antigen

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8⁺ T cell epitope, NY-ESO-1_88–96 (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1_157–165 epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1_88–96 is much more efficiently cross-presented from the soluble form, than NY-ESO-1_157–165. On the other hand, NY-ESO-1_157–165 is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A_26–35; whereas NY-ESO-1_88–96 was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1_88–96 is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18⁺ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1_88–96 from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8⁺ T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.     

Antibodies to gp120 and PD-1 Expression on Virus-Specific CD8+ T Cells in Protection from Simian AIDS

We compared the relative efficacies against simian immunodeficiency virus (SIV) challenge of three vaccine regimens that elicited similar frequencies of SIV-specific CD4⁺ and CD8⁺ T-cell responses but differed in the level of antibody responses to the gp120 envelope protein. All macaques were primed with DNA plasmids expressing SIV gag, pol, env, and Retanef genes and were boosted with recombinant modified vaccinia Ankara virus (MVA) expressing the same genes, either once (1 × MVA) or twice (2 × MVA), or were boosted once with MVA followed by a single boost with replication-competent adenovirus (Ad) type 5 host range mutant (Ad5 h) expressing SIV gag and nef genes but not Retanef or env (1 × MVA/Ad5). While two of the vaccine regimens (1 × MVA and 1 × MVA/Ad5) protected from high levels of SIV replication only during the acute phase of infection, the 2 × MVA regimen, with the highest anti-SIV gp120 titers, protected during the acute phase and transiently during the chronic phase of infection. Mamu-A*01 macaques of this third group exhibited persistent Gag CD8⁺CM9⁺ effector memory T cells with low expression of surface Programmed death-1 (PD-1) receptor and high levels of expression of genes associated with major histocompatibility complex class I (MHC-I) and MHC-II antigen. The fact that control of SIV replication was associated with both high titers of antibodies to the SIV envelope protein and durable effector SIV-specific CD8⁺ T cells suggests the hypothesis that the presence of antibodies at the time of challenge may increase innate immune recruiting activity by enhancing antigen uptake and may result in improvement of the quality and potency of secondary SIV-specific CD8⁺ T-cell responses.     

Acetyl CoA Carboxylase 2 Is Dispensable for CD8+ T Cell Responses

Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8⁺ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2) regulates fatty acid oxidation (FAO) by inhibiting carnitine palmitoyltransferase 1 (CPT1), a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8⁺ T cells, but does not appear to influence antigen-specific effector and memory CD8⁺ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8⁺ T cell responses.