CDK4/6 Inhibition Sensitizes Intracranial Tumors to PD-1 Blockade in Preclinical Models of Brain Metastasis.

PURPOSE: Brain metastases are associated with high morbidity and are often resistant to immune checkpoint inhibitors. We evaluated whether CDK4/6 inhibitor (CDKi) abemaciclib can sensitize intracranial tumors to programmed cell death protein 1 (PD-1) inhibition in mouse models of melanoma and breast cancer brain metastasis. EXPERIMENTAL DESIGN: Treatment response was evaluated in vivo using immunocompetent mouse models of brain metastasis bearing concurrent intracranial and extracranial tumors. Treatment effect on intracranial and extracranial tumor-immune microenvironments (TIME) was evaluated using immunofluorescence, multiplex immunoassays, high-parameter flow cytometry, and T-cell receptor profiling. Mice with humanized immune systems were evaluated using flow cytometry to study the effect of CDKi on human T-cell development. RESULTS: We found that combining abemaciclib with PD-1 inhibition reduced tumor burden and improved overall survival in mice. The TIME, which differed on the basis of anatomic location of tumors, was altered with CDKi and PD-1 inhibition in an organ-specific manner. Combination abemaciclib and anti-PD-1 treatment increased recruitment and expansion of CD8+ effector T-cell subsets, depleted CD4+ regulatory T (Treg) cells, and reduced levels of immunosuppressive cytokines in intracranial tumors. In immunodeficient mice engrafted with human immune systems, abemaciclib treatment supported development and maintenance of CD8+ T cells and depleted Treg cells. CONCLUSIONS: Our results highlight the distinct properties of intracranial and extracranial tumors and support clinical investigation of combination CDK4/6 and PD-1 inhibition in patients with brain metastases. See related commentary by Margolin, p. 257.

Virus-induced natural killer cell lysis of T cell subsets.

In addition to direct anti-viral activity, NK cells regulate viral pathogenesis by virtue of their cytolytic attack on activated CD4 and CD8 T cells. To gain insight into which differentiated T cell subsets are preferred NK targets, transgenic T cells were differentiated in vitro into Th0, Th1, Th2, Th17, Treg, Tc1, and Tc2 effector cells and then tested for lysis by enriched populations of lymphocytic choriomeningitis virus (LCMV)-induced activated NK cells. There was a distinct hierarchy of cytotoxicity in vitro and in vivo, with Treg, Th17, and Th2 cells being more sensitive and Th0 and Th1 cells more resistant. Some distinctions between in vitro vs in vivo generated T cells were explainable by type 1 interferon induction of class 1 histocompatibility antigens on the effector T cell subsets. NK receptor (NKR)-deficient mice and anti-NKR antibody studies identified no one essential NKR for killing, though there could be redundancies.

Weak vaccinia virus-induced NK cell regulation of CD4 T cells is associated with reduced NK cell differentiation and cytolytic activity.

Natural killer (NK) cells control antiviral adaptive immune responses in mice during some virus infections, but the universality of this phenomenon remains unknown. Lymphocytic choriomeningitis virus (LCMV) infection of mice triggered potent cytotoxic activity of NK cells (NKLCMV) against activated CD4 T cells, tumor cells, and allogeneic lymphocytes. In contrast, NK cells activated by vaccinia virus (VACV) infection (NKVACV) exhibited weaker cytolytic activity against each of these target cells. Relative to NKLCMV cells, NKVACV cells exhibited a more immature (CD11b-CD27+) phenotype, and lower expression levels of the activation marker CD69, cytotoxic effector molecules (perforin, granzyme B), and the transcription factor IRF4. NKVACV cells expressed higher levels of the inhibitory molecule NKG2A than NKLCMV cells. Consistent with this apparent lethargy, NKVACV cells only weakly constrained VACV-specific CD4 T-cell responses. This suggests that NK cell regulation of adaptive immunity, while universal, may be limited with viruses that poorly activate NK cells.

Modulation of let-7 miRNAs controls the differentiation of effector CD8 T cells.

The differentiation of naive CD8 T cells into effector cytotoxic T lymphocytes upon antigen stimulation is necessary for successful antiviral, and antitumor immune responses. Here, using a mouse model, we describe a dual role for the let-7 microRNAs in the regulation of CD8 T cell responses, where maintenance of the naive phenotype in CD8 T cells requires high levels of let-7 expression, while generation of cytotoxic T lymphocytes depends upon T cell receptor-mediated let-7 downregulation. Decrease of let-7 expression in activated T cells enhances clonal expansion and the acquisition of effector function through derepression of the let-7 targets, including Myc and Eomesodermin. Ultimately, we have identified a novel let-7-mediated mechanism, which acts as a molecular brake controlling the magnitude of CD8 T cell responses.

Heterologous Immunity and Persistent Murine Cytomegalovirus Infection.

One's history of infections can affect the immune response to unrelated pathogens and influence disease outcome through the process of heterologous immunity. This can occur after acute viral infections, such as infections with lymphocytic choriomeningitis virus (LCMV) and vaccinia virus, where the pathogens are cleared, but it becomes a more complex issue in the context of persistent infections. In this study, murine cytomegalovirus (MCMV) was used as a persistent infection model to study heterologous immunity with LCMV. If mice were previously immune to LCMV and then infected with MCMV (LCMV+MCMV), they had more severe immunopathology, enhanced viral burden in multiple organs, and suppression of MCMV-specific T cell memory inflation. MCMV infection initially reduced the numbers of LCMV-specific memory T cells, but continued MCMV persistence did not further erode memory T cells specific to LCMV. When MCMV infection was given first (MCMV+LCMV), the magnitude of the acute T cell response to LCMV declined with age though this age-dependent decline was not dependent on MCMV. However, some of these MCMV persistently infected mice with acute LCMV infection (7 of 36) developed a robust immunodominant CD8 T cell response apparently cross-reactive between a newly defined putative MCMV epitope sequence, M57727-734, and the normally subdominant LCMV epitope L2062-2069, indicating a profound private specificity effect in heterologous immunity between these two viruses. These results further illustrate how a history of an acute or a persistent virus infection can substantially influence the immune responses and immune pathology associated with acute or persistent infections with an unrelated virus. IMPORTANCE: This study extends our understanding of heterologous immunity in the context of persistent viral infection. The phenomenon has been studied mostly with viruses such as LCMV that are cleared, but the situation can be more complex with a persistent virus such as MCMV. We found that the history of LCMV infection intensifies MCMV immunopathology, enhances MCMV burden in multiple organs, and suppresses MCMV-specific T cell memory inflation. In the reverse infection sequence, we show that some of the long-term MCMV-immune mice mount a robust CD8 T cell cross-reactive response between a newly defined putative MCMV epitope sequence and a normally subdominant LCMV epitope. These results further illustrate how a history of infection can substantially influence the immune responses and immune pathology associated with infections with an unrelated virus.

Generation of cellular immune memory and B-cell immunity is impaired by natural killer cells.

The goal of most vaccines is the induction of long-lived memory T and B cells capable of protecting the host from infection by cytotoxic mechanisms, cytokines and high-affinity antibodies. However, efforts to develop vaccines against major human pathogens such as HIV and HCV have not been successful, thereby highlighting the need for novel approaches to circumvent immunoregulatory mechanisms that limit the induction of protective immunity. Here, we show that mouse natural killer (NK) cells inhibit generation of long-lived virus-specific memory T- and B cells as well as virus-specific antibody production after acute infection. Mechanistically, NK cells suppressed CD4 T cells and follicular helper T cells (T(FH)) in a perforin-dependent manner during the first few days of infection, resulting in a weaker germinal centre (GC) response and diminished immune memory. We anticipate that innovative strategies to relieve NK cell-mediated suppression of immunity should facilitate development of efficacious new vaccines targeting difficult-to-prevent infections.

MHC basis of T cell-dependent heterologous immunity to arenaviruses.

Having a history of infection with one pathogen may sometimes provide a level of T cell-dependent protective heterologous immunity to another pathogen. This immunity was initially thought due to cross-reactive T cell epitopes, but recent work has suggested that such protective immunity can be initiated nonspecifically by the action of cytokines on memory T cells. We retested this concept using two small and well-defined arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV), and found that heterologous immunity in these systems was indeed linked to T cell epitopes and the major histocompatibility complex.

Therapeutic depletion of natural killer cells controls persistent infection.

Persistent viral infections are associated with host and viral factors that impair effective antiviral immunity. Natural killer (NK) cells contribute to establishment of persistent lymphocytic choriomeningitis virus (LCMV) infection in mice through suppression of virus-specific T cell responses during the first few days of infection, but NK cell depletion during those early time points can enable severe T cell-mediated immune pathology and death of the host. Here we show that long after their peak in cytolytic activation, NK cells continue to support viral persistence at later times of infection. Delayed depletion of NK cells, 2 to 3 weeks after infection, enhanced virus-specific T cell responses and viral control. This enhancing effect of delayed NK cell depletion on antiviral immunity, in contrast to early NK cell depletion, was not associated with increased morbidity and mortality, and mice quickly regained weight after treatment. The efficacy of the depletion depended in part upon the size of the original virus inoculum, the viral load at the time of depletion, and the presence of CD4 T cells. Each of these factors is an important contributor to the degree of CD8 T cell dysfunction during viral persistence. Thus, NK cells may continuously contribute to exhaustion of virus-specific T cells during chronic infection, possibly by depleting CD4 T cells. Targeting of NK cells could thus be considered in combination with blockade of other immunosuppressive pathways, such as the interleukin-10 (IL-10) and programmed death 1 (PD-1) pathways, as a therapy to cure chronic human infections, including those with HIV or hepatitis C virus. IMPORTANCE Persistent virus infections are a major threat to global human health. The capacity of viruses, including HIV and hepatitis C virus, to overwhelm or subvert host immune responses contributes to a prolonged state of dampened antiviral immune functionality, which in turn facilitates viral persistence. Recent efforts have focused on therapeutics that can restore the effector functions of these functionally exhausted virus-specific T cells in order to expedite viral clearance. Here we establish that natural killer (NK) cells actively contribute to immune dysfunction and viral persistence at later stages of infection. This previously undescribed mechanism of immune suppression during chronic infection provides a vital clue for the design of novel therapeutic strategies targeting NK cell immunosuppressive activity in order to restore immune function and enhance viral control in chronically infected individuals.

Disparate epitopes mediating protective heterologous immunity to unrelated viruses share peptide-MHC structural features recognized by cross-reactive T cells.

Closely related peptide epitopes can be recognized by the same T cells and contribute to the immune response against pathogens encoding those epitopes, but sometimes cross-reactive epitopes share little homology. The degree of structural homology required for such disparate ligands to be recognized by cross-reactive TCRs remains unclear. In this study, we examined the mechanistic basis for cross-reactive T cell responses between epitopes from unrelated and pathogenic viruses, lymphocytic choriomeningitis virus (LCMV) and vaccinia virus. Our results show that the LCMV cross-reactive T cell response toward vaccinia virus is dominated by a shared asparagine residue, together with other shared structural elements conserved in the crystal structures of K(b)-VV-A11R and K(b)-LCMV-gp34. Based on analysis of the crystal structures and the specificity determinants for the cross-reactive T cell response, we were able to manipulate the degree of cross-reactivity of the T cell response, and to predict and generate a LCMV cross-reactive response toward a variant of a null OVA-derived peptide. These results indicate that protective heterologous immune responses can occur for disparate epitopes from unrelated viruses.

Alloreactive CD8 T cells rescued from apoptosis during co-stimulation blockade by Toll-like receptor stimulation remain susceptible to Fas-induced cell death.

Blockade of co-stimulatory signals to T cells is extremely effective for the induction of transplantation tolerance in immunologically naive rodents. However, infections and inflammation compromise the efficacy of co-stimulation blockade regimens for the induction of tolerance, thereby stimulating the rejection of allografts. Previous studies have shown that stimulation of innate immunity abrogates tolerance induction by preventing the deletion of alloreactive CD8(+) T cells that normally occurs during co-stimulation blockade. Although inflammation prevents the deletion of alloreactive T cells during co-stimulation blockade, it is not known if this resistance to cell death is the result of a mechanism intrinsic to the T cell. Here, we used syngeneic bone marrow chimeric mice that contain a trace population of T-cell receptor transgenic alloreactive CD8(+) T cells to investigate the early apoptotic signature and activation status of alloreactive T cells following exposure to inflammatory signals during co-stimulation blockade with an antibody specific for CD154. Our findings revealed that the presence of bacterial lipopolysaccharide during co-stimulation blockade enhanced the early activation of alloreactive CD8(+) T cells, as indicated by the up-regulation of CD25 and CD69, suppressed Fas ligand expression, and prevented apoptotic cell death. However, alloreactive CD8(+) T cells from lipopolysaccharide-treated mice remained sensitive to Fas-mediated apoptosis in vitro. These findings suggest that alloreactive T cells rescued from deletion during co-stimulation blockade by inflammation are still sensitive to pro-apoptotic signals and that stimulating these apoptotic pathways during co-stimulation blockade may augment the induction of tolerance.

Bi-specific MHC heterodimers for characterization of cross-reactive T cells.

T cell cross-reactivity describes the phenomenon whereby a single T cell can recognize two or more different peptide antigens presented in complex with MHC proteins. Cross-reactive T cells have previously been characterized at the population level by cytokine secretion and MHC tetramer staining assays, but single-cell analysis is difficult or impossible using these methods. In this study, we describe development of a novel peptide-MHC heterodimer specific for cross-reactive T cells. MHC-peptide monomers were independently conjugated to hydrazide or aldehyde-containing cross-linkers using thiol-maleimide coupling at cysteine residues introduced into recombinant MHC heavy chain proteins. Hydrazone formation provided bi-specific MHC heterodimers carrying two different peptides. Using this approach we prepared heterodimers of the murine class I MHC protein H-2K(b) carrying peptides from lymphocytic choriomeningitis virus and vaccinia virus, and used these to identify cross-reactive CD8+ T cells recognizing both lymphocytic choriomeningitis virus and vaccinia virus antigens. A similar strategy could be used to develop reagents to analyze cross-reactive T cell responses in humans.

High frequencies of virus-specific CD8+ T-cell precursors.

A productive CD8(+) T-cell response to a viral infection requires rapid division and proliferation of virus-specific CD8(+) T cells. Tetramer-based enrichment assays have recently given estimates of the numbers of peptide-major histocompatibility complex-specific CD8(+) T cells in naïve mice, but precursor frequencies for entire viruses have been examined only by using in vitro limiting-dilution assays (LDAs). To examine CD8(+) T-cell precursor frequencies for whole viruses, we developed an in vivo LDA and found frequencies of naïve CD8(+) T-cell precursors of 1 in 1,444 for vaccinia virus (VV) ( approximately 13,850 VV-specific CD8(+) T cells per mouse) and 1 in 2,958 for lymphocytic choriomeningitis virus (LCMV) ( approximately 6,761 LCMV-specific CD8(+) T cells per mouse) in C57BL/6J mice. In mice immune to VV, the number of VV-specific precursors, not surprisingly, dramatically increased to 1 in 13 ( approximately 1,538,462 VV-specific CD8(+) T cells per mouse), consistent with estimates of VV-specific memory T cells. In contrast, precursor numbers for LCMV did not increase in VV-immune mice (1 in 4,562, with approximately 4,384 LCMV-specific CD8(+) T cells per VV-immune mouse). Using H-2D(b)-restricted LCMV GP33-specific P14-transgenic T cells, we found that, after donor T-cell take was accounted for, approximately every T cell transferred underwent a full proliferative expansion in response to LCMV infection. This high efficiency was also seen with memory populations, suggesting that most antigen-specific T cells will proliferate extensively at a limiting dilution in response to infections. These results show that frequencies of naïve and memory CD8(+) T cell precursors for whole viruses can be remarkably high.

Rapid quantification of naive alloreactive T cells by TNF-alpha production and correlation with allograft rejection in mice.

Allograft transplantation requires chronic immunosuppression, but there is no effective strategy to evaluate the long-term maintenance of immunosuppression other than assessment of graft function. The ability to monitor naive alloreactive T cells would provide an alternative guide for drug therapy at early, preclinical stages of graft rejection and for evaluating tolerance-inducing protocols. To detect and quantify naive alloreactive T cells directly ex vivo, we used the unique ability of naive T cells to rapidly produce TNF-alpha but not IFN-gamma. Naive alloreactive T cells were identified by the production of TNF-alpha after a 5-hour in vitro stimulation with alloantigen and were distinguished from effector/memory alloreactive T cells by the inability to produce IFN-gamma. Moreover, naive alloreactive T cells were not detected in mice tolerized against specific alloantigens. The frequency of TNF-alpha-producing cells was predictive for rejection in an in vivo cytotoxicity assay and correlated with skin allograft rejection. Naive alloreactive T cells were also detected in humans, suggesting clinical relevance. We conclude that rapid production of TNF-alpha can be used to quantify naive alloreactive T cells, that it is abrogated after the induction of tolerance, and that it is a potential tool to predict allograft rejection.

Rapid production of TNF-alpha following TCR engagement of naive CD8 T cells.

The acquisition of effector functions by naive CD8 T cells following TCR engagement is thought to occur sequentially with full functionality being gained only after the initiation of division. We show that naive CD8 T cells are capable of immediate effector function following TCR engagement, which stimulates the rapid production of TNF-alpha. Stimulation of splenocytes from naive mice of differing genetic backgrounds with anti-CD3epsilon mAb resulted in significant production of TNF-alpha by naive CD8 T cells within 5 h. Moreover, naive lymphocytic choriomeningitis virus-specific TCR-transgenic CD8 T cells stimulated with either their cognate peptide ligand or virus-infected cells produced TNF-alpha as early as 2 h poststimulation, with production peaking by 4 h. Naive CD8 T cells produced both membrane-bound and soluble TNF-alpha. Interfering with TNF-alpha activity during the initial encounter between naive CD8 T cells and Ag loaded dendritic cells altered the maturation profile of the APC and diminished the overall viability of the APC population. These findings suggest that production of TNF-alpha by naive CD8 T cells immediately after TCR engagement may have an unappreciated impact within the local environment where Ag presentation is occurring and potentially influence the development of immune responses.

Rapid conversion of effector mechanisms from NK to T cells during virus-induced lysis of allogeneic implants in vivo.

Viral infections can strongly stimulate both NK cell and allospecific CD8 T cell responses, and these same effector cells can lyse allogeneic cell lines in vitro. However, the impact of viral infections on the effector systems mediating rejection of allogeneic tissues in vivo has not been fully explored. Using in vivo cytotoxicity assays, we evaluated the effector systems mediating the rejection of CFSE-labeled allogeneic splenocytes after an infection of C57BL/6 (B6) mice with lymphocytic choriomeningitis virus. Naive B6 mice predominantly used a NK cell-effector mechanism to reject allogeneic splenocytes because they rejected BALB/C (H2(d)) splenocytes but not CBA (H2(k)) splenocytes, and the rejection was prevented by immunodepletion of NK1.1(+) or Ly49D(+) NK cells. This rapid and efficient in vivo cytotoxicity assay recapitulated the specificity of NK cell-mediated rejection seen in longer duration in vivo assays. However, as early as 1 day after infection with lymphocytic choriomeningitis virus, a CD8 T cell-dependent mechanism participated in the rejection process and a broader range of tissue haplotypes (e.g., H2(k)) was susceptible. The CD8 T cell-mediated in vivo rejection process was vigorous at a time postinfection (day 3) when NK cell effector functions are peaking, indicating that the effector systems used in vivo differed from those observed with in vitro assays measuring the killing of allogeneic cells. This rapid generation of allospecific CTL activity during a viral infection preceded the peak of viral epitope-specific T cell responses, as detected by in vivo or in vitro cytotoxicity assays.

Direct visualization of cross-reactive effector and memory allo-specific CD8 T cells generated in response to viral infections.

CD8 T cell cross-reactivity between heterologous viruses has been shown to provide protective immunity, induce immunopathology, influence the immunodominance of epitope-specific T cell responses, and shape the overall memory population. Virus infections also induce cross-reactive allo-specific CTL responses. In this study, we quantified the allo-specific CD8 T cells elicited by infection of C57BL/6 (B6) mice with lymphocytic choriomeningitis virus (LCMV). Cross-reactive LCMV-specific CD8 T cells were directly visualized using LCMV peptide-charged MHC tetramers to costain T cells that were stimulated to produce intracellular IFN-gamma in response to allogeneic target cells. The cross-reactivity between T cells specific for LCMV and allogeneic Ags was broad-based, in that it involved multiple LCMV-derived peptides, but there were distinctive patterns of reactivity against allogeneic cells with different haplotypes. Experiments indicated that this cross-reactivity was not due to the expression of two TCR per cell, and that the patterns of allo-reactivity changed during sequential infection with heterologous viruses. The allo-specific CD8 T cells generated by LCMV infection were maintained at relatively high frequencies in the memory pool, indicating that memory allo-specific CD8 T cell populations can arise as a consequence of viral infections. Mice previously infected with LCMV and harboring allo-specific memory T cells were refractory to the induction of tolerance to allogeneic skin grafts.

T cell immunodominance and maintenance of memory regulated by unexpectedly cross-reactive pathogens.

We show here that T cell cross-reactivity between heterologous viruses influences the immunodominance of virus-specific CD8(+) T cells by two mechanisms. First, T cells specific for cross-reactive epitopes dominate acute responses to viral infections; second, within the memory pool, T cells specific for cross-reactive epitopes are maintained while those specific for non-cross-reactive epitopes are selectively lost. These findings suggest an immunological paradigm in which viral infections shape the available T cell repertoire, causing alterations in the hierarchies of both the primary and memory CD8(+) T cell responses elicited by subsequent viral infections. Thus, immunodominance is a function of the host's previous exposure to unrelated pathogens, and this may have an impact on protective immunity and immunopathology.