CD8 T Cell Memory Increases Immunopathology in the Perforin-Deficient Model of Hemophagocytic Lymphohistiocytosis Secondary to TNF-α

Abstract

Familial hemophagocytic lymphohistiocytosis 2 (FHL2) is a cytokine storm syndrome characterized by immune hyperactivation with viral infection due to a CD8 T cell cytotoxic killing defect secondary to a perforin deficiency. As most studies of FHL2 mice have used pathogen naïve animals, the effects of immune memory on FHL2 are understudied. We utilized an immunization model of the perforin-deficient mouse to study the effects of immune memory on FHL2. Prior CD8 T cell specific antigen exposure leads to enhanced HLH disease with increased morbidity and decreased time to mortality. Enhanced disease is associated with altered cytokine production and T cell proliferation. Response to IFNγ blockade is reduced and TNFα gains a pathogenic role, while blockade of the IL-33 receptor ST2 remains effective. These results suggest that pre-existing immune memory may worsen outcome and alter treatment response for FHL2 patients who may not be naïve to their immune triggers.

Introduction

Familial hemophagocytic lymphohistiocytosis type 2 (FHL2) is a classic genetic form of HLH caused by mutations within the perforin gene that limit cytolytic elimination of viral infections by CD8 T cells and NK cells.(1) This disease affects infants early in life and has a high mortality rate.(2, 3) Currently, the only viable treatment option for FHL2 is immuno-chemotherapy followed by bone marrow transplant,(3, 4) but other biologically directed therapies, including interferon gamma (IFNγ) blockade, have demonstrated effectiveness in mouse models(5-8) and are being tested in on-going clinical trials (ClinicalTrials.gov Identifier: NCT01818492).(9)

The murine model of FHL2 uses the perforin-deficient mouse (Prf1^−/−) on the C57BL/6 (B6) genetic background.(8) In this model, FHL is triggered by the lymphocytic choriomeningitis virus (LCMV), a virus that would normally be cleared by the CD8 T cell cytolytic response in perforin sufficient mice. Prf1^−/− mice develop lethal HLH secondary to CD8 T cell-mediated IFNγ production, as CD8 T cell depletion or IFNγ neutralization protect Prf1^−/− mice from disease. We have additionally shown that blockade of the IL-33 receptor ST2 is also able to prevent disease and lower IFNγ levels.(5) However, other cytokines including TNFα do not contribute to disease progression.(8)

All previous studies of the B6 Prf1^−/− murine model of FHL2 have been performed in immunologically naïve mice. A previous study by Badovinac et al. demonstrated that unlike B6 Prf1^−/− mice, naïve BALB/c Prf1^−/− mice did not develop mortality upon LCMV infection. However, immunization with CD8 T-cell, LCMV specific peptides prior to infection rendered these mice susceptible to an IFNγ-dependent mortality similar to FHL2.(10) Unlike the BALB/c Prf1^−/− mouse, the B6 Prf1^−/− mouse develops HLH disease on first exposure to the virus without the need for prior immunization. Although the Badovinac study does mention that immunization of B6 Prf1^−/− mice followed by LCMV infection results mortality with LCMV specific CD8 T-cell expansion (as do naïve B6 Prf1^−/− mice), these data were not shown as the focus of the report was on the Balb/c model. Neither was a full immunologic dissection or comparision to unimmunized mice presented in the B6 model.(10) We therefore questioned whether the naïve, predisposed B6 Prf1^−/− model would develop alterations in disease upon immunization prior to infection, much like the Balb/c model switches from resistant to permissive with immunization. Humans with FHL2 may not have a truly naïve immune response to their viral trigger, as they could possess a pre-existing memory T cell response to an HLH trigger without prior exposure to this specific trigger. Given the integral role of CD8 T cells in FHL2 and the known differences in the immune response of memory CD8 T cells compared to naïve T cells, we hypothesized that CD8 T cell memory may act as an important modifier of FHL2.

Materials and Methods

Mice

C57BL/6 (WT) and perforin-deficient (C57BL/6-Prf1^tm1Sdz/J, Prf1^−/−) mice were obtained from Jackson Laboratory and bred in our specific-pathogen free barrier facility. All animal studies were performed according to The Children’s Hospital of Philadelphia Institutional Animal Care and Use Committee.

In Vivo Immunization

A previously published protocol for peptide-coated dendritic cell immunization was adapted for this study.(11) Briefly, Flt3L induced splenic DCs were isolated with CD11c magnetic beads and pulsed with LCMV peptides (2 γM) for two hours. GP33 and NP396 purified peptide were obtained from Genscript. 2.5 × 10⁵ DCs were injected retro-orbitally into 4-8 week Prf1^−/− mice. Control mice were injected with equal numbers of unpulsed DCs.

FHL2 Induction

Mice between the ages of 8-12 weeks were injected intraperitoneally with 2×10⁵ plaque-forming units of LCMV-Armstrong strain. Mice were euthanized if they developed significant morbidity or excessive weight loss (>20% total body weight).. Viral titers were measured using plaque assay with Vero cells as previously described.(12)

Organ weights and histology

Spleen and liver weights were obtained from mice sacrificed on day 7 post-infection. For pathologic analysis, organs were fixed overnight in 4% paraformaldehyde, imbedded in paraffin and stained with hematoxylin and eosin. Images were obtained and lymphocytic nodules were quantified as average number per high power field, three fields sampled.

Flow cytometric analysis

Splenocytes were obtained following single cell suspension and red blood cell lysis. Staining was performed with LIVE/DEAD fixable viability dye (Life Technologies) and CD4, CD8α, CD44, CD90.2, IFNγ, and TNFα (BD Bioscience, Biolegend). The H-2D^bGP_33-41 major histocompatibility complex-peptide tetramer was used for antigen-specific CD8 T cell identification and were provided as fluorophore-conjugated tetramers by E.J.W. All samples were acquired on MACSQuant flow cytometer (Miltenyi Biotec) and analyzed using FlowJo software version 10.

Peptide restimulation assay

10⁶ splenocytes were cultured without peptide or with 0.2 μM LCMV GP33 peptide, NP396 peptide for five hours. IFNγ positivity by flow cytometry was used to identify LCMV specific cells.(13)

Statistical analysis

Weight loss data was analyzed by linear mixed-effects models using R (R Core Team, 2014). Survival statistical analysis was performed by log-rank Mantel-Cox test. GraphPad Prism 5 was used for statistical testing and p-values were calculated using two-way Student’s T test or 2-way ANOVA when appropriate.

In vivo blockade

Rat anti-mouse ST2-blocking antibody (ST2 blocking antibody) and mouse IgG1 isotype control antibody were provided by Amgen. LCMV-infected mice were injected intraperitoneally with 150 μg ST2 blocking antibody or isotype control every other day beginning on day 2 post-infection.(14) All other neutralizing and isotype control antibodies were obtained from BioXcell. Rat anti-mouse IFNγ (XMG1.2, 0.5 mg), rat anti-mouse TNFα (XT3.11, 1 mg), and rat IgG2a isotype control (2A3, 0.5 mg) were injected intraperitoneally every third day beginning on day 2 post-infection.(8)

Results

CD8 T-cell memory increases clinical markers, symptoms, and HLH disease severity in Prf1^−/− mice

We first examined differences between B6 Prf1^−/− HLH responses in naïve mice and mice immunized against the CD8 LCMV epitopes GP33 and NP396 using a well-characterized dendritic cell immunization protocol(15). Control mice were treated with the same immunization protocol without peptide exposure. Thirty days after priming the CD8 T cell memory response, we compared the development of HLH in immunized and control mice prior to LCMV infection. Immunization with GP33 loaded dendritic cells led to the development of significant numbers CD8+ T-cells capable of making rapid IFNγ responses to GP33 restimulation, consistent with a memory response, that was absent in control immunized mice. (Figure 1A) Similar development of rapidly responding peptide specific CD8 memory T cells was seen with NP396 peptide, confirming this immunization approach with multiple peptides (data not shown).

Figure 1. Prf1^−/− mice exhibit increased immunopathology with prior CD8 T cell memory secondary to immunization.

Prf1^−/− mice were immunized with GP33, NP396, or control dendritic cells, rested for 30 days and either sacrificed prior to infection or infected with LCMV. Following LCMV infection, mice were sacrificed on day 7 post-infection or monitored for survival. A. Prior to infection, total IFNγ producing GP33 specific CD8 T cells per spleen (CD90, CD8, CD44, IFNγ) as represented by IFNγ production in response to GP33 peptide restimulation in immunized and control mice (n=3). B. Survival of GP33 and NP396 CD8 T cell immunized mice with LCMV infection following 30 day rest period compared to control mice. Analyzed by log-rank Mantel-Cox test. * - P<0.05 for control compared to both immunization groups (n=5-6 per group). C. Percent body mass weight loss of GP33 immunized Prf1^−/− mice versus control. * - P<0.05 by linear mixed effects modeling (n=5 per group). D. Comparison of hematologic parameters (n=5-7) on day 7 post-infection with LCMV. E. Comparison of lymphocytic nodular infiltration (Lymphocytic nodules per HPF) in the liver in GP33 immunized or control mice and representative sections (n=6-8). F. Comparison of ferritin (n=9-10) and sCD25/IL2R (n=9-12) in serum on day 7 post-infection with LCMV. *P<0.05 by 2-way Student’s T-test except as indicated. All experiments represent at minimum two independent experiments.

Following infection and triggering of HLH, immunization led to significantly increased mortality. For GP33 immunized mice, average survival after LCMV infection decreased from 14 days in control mice to 9 days in immunized mice (Figure 1B). A similar decrease in time to mortality was seen following NP396 immunization (Figure 1B), indicating that increased disease severity occurs with CD8 T cell memory against multiple epitope specificities. Immunization-enhanced HLH was associated with significant increases in other markers of HLH disease activity including accelerated weight loss (Figure 1C), and more severe anemia and thrombocytopenia (Figure 1D). As there were no differences in the enhanced response comparing GP33 immunization to NP396 immunization (data not shown), all further experiments focus on the GP33 immunization. Hepatitis was markedly enhanced in immunized Prf1^−/− mice compared to control mice, with significantly more inflammatory lobular infiltrates (Figure 1E). Also consistent with increased disease severity, GP33 immunized mice had elevation of serum markers of HLH, including significantly elevated ferritin and a trend toward elevated sCD25/IL2Ra (Figure 1F). These results demonstrate that GP33 immunization and resultant CD8 T cell LCMV directed memory enhance LCMV-induced HLH in B6 Prf1^−/− mice.

Enhanced HLH disease is associated with enhanced expansion and function of peptide-specific CD8 T cells post-infection

To characterize the immune milieu that correlated with immunization-enhanced HLH, mice were sacrificed on day 7 post-infection. Serum levels of IFNγ were significantly elevated in immunized mice compared to controls. Serum levels of the pro-inflammatory cytokines IL-6 and TNFα were unchanged in immunized mice, while the anti-inflammatory cytokine IL-10 was significantly depressed (Figure 2A). To characterize the peptide specific CD8 immune response, spleen cells were obtained on day 7 post-infection and stimulated with GP33 peptide, NP396 peptide, or no peptide. In infected mice, absolute numbers of splenic GP33 specific CD8 T cells in immunized mice were increased 6-fold compared to control mice as shown by IFNγ production with peptide stimulation (Figure 2B). Tetramer staining for GP33-specific CD8 T cells in LCMV-infected mice corroborated this dramatic increase in GP33-specific T cells in immunized mice compared to naïve mice (Figure 2B, Supplemental Figure 1A). In GP33 immunized mice, GP33 specific CD8 T cells represented almost 45% of total CD8+ T cells compared to approximately only 8% in naïve mice (Figure 2C, Supplemental Figure 1B). Immunization with GP33 did not result in expansion of unrelated LCMV epitope specific T cells after infection, as evidenced by the lack of expansion of NP396 specific cells in GP33 immunized mice compared to naïve mice (Figure 2C, Supplemental Figure 1B). Thus, the increased pathology seen in immunization-enhanced HLH is associated with expansion of CD8 T cells specific to the peptide used for immunization. Immunization increased the frequency and number of GP33-specific, TNFα-producing splenic CD8 T cells at day 7 post-infection by 3-4 fold as measured by GP33 peptide restimulation (Figure 2D, Supplemental Figure 1C). Importantly, splenic titers of LCMV measured on day 7 post-infection are unchanged by immunization indicating that, despite functional enhancement in the CD8 T cell immune response, virus is still not cleared by perforin-deficient mice that have been immunized (Figure 2E).

Figure 2. CD8 T-cell memory alters the immune profile in enhanced HLH without altering viral load.

Prf1^−/− mice were immunized against GP33 or control, rested for 30 days and infected with LCMV. Mice were sacrificed on day 7 post-infection, serum samples were obtained and splenocytes were stimulated in vitro and analyzed. A. Serum cytokine levels in immunized and control mice (n=8-12). B. Total GP33-specific CD8 T cells (CD90+, CD8+, CD44+, IFNγ+) in immunized and control mice determined by IFNγ production following in vitro stimulation (n=6-8). Total number of GP33-specific CD8 T cells determined by GP33 tetramer staining (CD90+, CD8+, CD44+, GP33 tetramer+) (n=6-7). C. Percent of total CD8 T cells specific for GP33 or NP396 determined by IFNγ production following in vitro peptide stimulation with the respective peptide in GP33 immunized and control mice on day 7 post-infection (CD90+, CD8+, CD44+, IFNγ+) (n=4). D. Seven days post LCMV infection, total number of GP33 specific TNFα producing splenic CD8 T cells (CD90+, CD8+, CD44+, TNFα+) following in vitro peptide stimulation (n=6-8). E. LCMV splenic titers (n=6-8). Log scale is shown. *P<0.05 by 2-way Student’s T-test. All plots representative of a minimum of two experimental replicates.

Immunization-enhanced HLH is partially ameliorated by IFNγ blockade and is fully dependent on ST2 receptor activation

IFNγ production is one of the primary effector functions of activated CD8 T cells in response to LCMV infection, and has been shown to be a key component of mortality in the naïve mouse model of FHL2.(8) IFNγ neutralization in LCMV-infected naïve Prf1^−/− mice protects from mortality, which supports the use of IFNγ neutralization in human HLH patients.(9) As IFNγ neutralization has only been tested in naïve mice on the B6 background, we questioned whether IFNγ blockade would remain effective in immunization-enhanced HLH disease. As previously reported, IFNγ blockade led to significant improvement in survival in unimmunized mice, but surprisingly in GP33-immunized mice, IFNγ blocked mice continued to have significant mortality, with an average survival of 15 days post-infection, similar to control mice without IFNγ blockade (Figure 3A). Importantly, these mice still had significant improvement in survival compared to immunized mice receiving placebo treatment (average survival 9 days), indicating that IFNγ continues to play an important role in mortality in enhanced HLH disease. However, these data argued that IFNγ is not the only component leading to the increased disease severity of enhanced HLH in immunized mice. We considered that the apparent loss of efficacy of IFNγ blockade in immunized mice might be explained by increased IFNγ production and therefore an underdosing of blocking antibody and incomplete IFNγ blockade. However, IFNγ blockade as assessed by expression of the IFNγ-inducible gene product MHCII on Ly6C^hi inflammatory monocytes was equally downregulated with IFNγ blockade in all groups of mice, suggesting that the pharmacodynamic effects of IFNγ blockade were comparable between control and immunized mice (Figure 3B). This indicates that inadequate dosing is not the explanation for the loss of efficacy. Collectively, our data suggest that IFNγ is not the only factor leading to increased disease severity in immunization-enhanced HLH.

Figure 3. CD8 T-cell memory alters HLH response to cytokine blockade.

A. Survival of GP33 immunized mice with LCMV infection following 30 day rest period compared to control mice with IFNγ blockade or isotype control antibody treatment. Mice were injected IP starting on day 2 and every 3 days thereafter. Analyzed by log-rank Mantel-Cox test. P<0.05, n=6 for unblocked groups, 9 for IFNγ blocked groups. B. GP33 immunized and control mice were treated with IFNγ blockade or isotype control antibody treatment. Mice were injected IP starting on day 2 and every 3 days thereafter and were sacrificed on day 7 post-infection. Inflammatory monocytes were analyzed for MHCII expression (CD90-, Ly6G-, CD11b+, Ly6Chi, MHCII) by flow cytometry with representative flow plots. Effect of IFNγ blockade is significant, with no significant effect of immunization status by 2-way ANOVA, *P<0.05, n=4 per group. C. Survival of GP33 immunized mice with LCMV infection following 30 day rest period compared to control mice with ST2 blockade or isotype control antibody treatment. Mice were injected IP starting on day 2 and every 2 days thereafter. Analyzed by log-rank Mantel-Cox test. P<0.05, n=5 for all groups. All plots represent minimum of two experimental replicates.

We have recently shown that blockade of the IL33 receptor ST2 leads to a decrease in the cytokine storm and enhanced survival in naïve FHL2 mice.(5) To determine if ST2 blockade would ameliorate disease in immunization-enhanced HLH, we treated LCMV-infected, GP33-immunized Prf1^−/− mice with ST2 blocking antibody. Unlike IFNγ blockade, which lost effectiveness in immunization-enhanced HLH, ST2 blockade remained equally effective in immunized and control mice, with equivalent survival in both immunized and control mice to up to 18 days post-infection (Figure 3C). These data suggest that ST2 controls both the IFNγ dependent and independent factors that contribute to immunization-enhanced HLH.

TNFα is an IFNγ-independent mediator of immunopathology in immunization-enhanced HLH

We observed that prior immunization resulted in an increase in the number of gp33-specific, TNFα-producing, splenic CD8+ T cells on day 7 post-infection (Figure 2D, Supplemental Figure 1D). We thus hypothesized that increases in TNFα production by memory CD8 T cells could be an IFNγ-independent mediator of HLH immunopathology during immunization-enhanced HLH. To this end, we blocked IFNγ and investigated the immunologic characteristics of immunized and control mice to investigate whether TNFα may gain importance. Serum TNFα was not significantly altered in the serum on day 7 post-infection in LCMV-infected GP33-immunized IFNγ-blocked mice (Figure 4A). Similarly, IFNγ blockade did not affect the expansion of GP33-specific IFNγ⁺ CD8 T cells (Figure 4B) or GP33-specific TNFα⁺ CD8 T cells (Figure 4C), though immunized mice continued to have an increased percentage of cytokine producing cells within the total CD8 T cell compartment. The total number of TNFα producing GP33-specific CD8 T cells in immunized mice was increased by approximately 4-fold, a difference that remained with IFNγ blockade (Figure 4D). Though there was no significant increase in serum TNFα levels, with the significant expansion of TNFα producing GP33 specific CD8 T cells, we hypothesized that TNFα may be playing an IFNγ-independent pathogenic role in immunization-enhanced HLH at the tissue level, a role that may emerge with IFNγ blockade. To test this hypothesis, TNFα and IFNγ were blocked in GP33-immunized mice to determine whether dual blockade would enhance survival in immunized mice. Consistent with our hypothesis, TNFα and IFNγ dual blockade led to a significant improvement in survival of LCMV-infected GP33-immunized mice, equaling the survival of naïve mice treated with IFNγ blockade alone (Figure 4E). Dual blockade did not result in any measurable increase in survival in naïve mice, suggesting the effect of TNFα was specific to the immunized scenario. Interestingly, TNFα blockade alone was insufficient to cause a change in survival in either the naïve or immunize mice, suggesting its effects can only be ascertained in the background of IFNγ blockade.

Figure 4. TNFα is produced by memory CD8 T cells and contributes to mortality in immune memory HLH.

Prf1^−/− mice were immunized against GP33 or with control procedure, rested for 30 days, infected with LCMV and treated with either IFNγ blockade or isotype control beginning day 2 post-infection, and every 3^rd day thereafter. Mice were sacrificed on day 7 post-infection. A. Serum TNFα shows no difference in immune memory compared to control and this is unaffected by IFNγ blockade. Not significant by 2-way ANOVA (n=10-14, pooled two experimental replicates). B. Percent GP33-specific memory CD8 T cells of total splenic CD8 T cells. Expansion is unaffected by IFNγ blockade (CD90, CD8, CD44, IFNγ) however the effect of immune memory is significant by 2-way ANOVA, *P<0.05. IFNγ blockade has no effect (n=3-4, two experimental replicates). C. Percent TNFα producing GP33-specific CD8 T cells of total splenic CD8 T cells. Expansion is unaffected by IFNγ blockade (CD90, CD8, CD44, TNFα) however the effect of immune memory is significant by 2-way ANOVA, *P<0.05 (n=3-4, two experimental replicates). D. Total number of splenic TNFα producing GP33 specific CD8 T cells in immunized and control mice with or without IFNγ blockade (CD90, CD8, CD44, TNFα). Effect of immune memory is significant by 2-way ANOVA, *P<0.05. IFNγ blockade has no effect (n=3-4, two experimental replicates). E. Mice were treated beginning day 2 post-infection, and every 3^rd day thereafter with either isotype control, IFNγ blockade only, TNFα blockade only, or IFNγ/TNFα dual blockade following infection. Left side panel shows control unimmunized mice, right side panel shows immunized mice. Analyzed by log-rank Mantel-Cox test. * = P<0.0001, Pooled experiments, n=5-15 mice per group.

Discussion

Taken together these results show that in FHL2 on the B6 permissive background, pre-existing CD8 T-cell memory results in an enhanced HLH disease. This parallels prior observations that on the resistant Balb/c background, pre-existing CD8 T-cell memory makes disease permissive.(10). However, unlike the Balb/c model, immunization in B6 mice results in a qualitative difference, with prominence of other cytokines in addition to IFNγ and specificially with TNFα gaining a pathogenic role as an effector cytokine. Interestingly, ST2 blockade retains its efficacy, suggesting that this is an upstream mediator of disease that is unaltered by memory status. Further work in human patients and in other mouse models of HLH will be needed to generalize these findings to other HLH syndromes beyond FHL2. However, these results suggest that there may exist immunologic scenarios where IFNγ blockade may not be sufficient to treat HLH and alternative biologic therapies may need to be considered.