CD8 T cell-mediated depletion of HBV surface-antigen-expressing, bilineal-differentiated liver carcinoma cells generates highly aggressive escape variants

The expression of viral antigens in chronic hepatitis B virus (HBV) infection drives continuous liver inflammation, one of the main risk factors to develop liver cancer. HBV developed immune-suppressive functions to escape from the host immune system, but their link to liver tumor development is not well understood. Here, we analyzed if and how HBV surface antigen (HBs) expression in combined hepatocellular-cholangiocarcinoma (cHCC/iCCA) cells influences their antigenicity for CD8 T cells. We randomly isolated liver tumor tissues from AlfpCre+-Trp53fl/fl/Alb-HBs+ tg mice and established primary carcinoma cell lines (pCCL) that showed a bilineal (CK7+/HNF4α+) cHCC/iCCA phenotype. These pCCL uniformly expressed HBs (HBshi), and low levels of MHC-I (MHC-Ilo), and were transiently convertible to a high antigenicity (MHC-Ihi) phenotype by IFN-γ treatment. HBshi/pCCL induced HBs/(Kb/S190–197)-specific CD8 T cells and developed slow-growing tumors in subcutaneously transplanted C57Bl/6J (B6) mice. Interestingly, pCCL-ex cells, established from HBshi/pCCL-induced and re-explanted tumors in B6 but not those in immune-deficient Rag1−/− mice showed major alterations, like an MHC-Ihi phenotype, a prominent growth-biased gene expression signature, a significantly decreased HBs expression (HBslo) and a switch to fast-growing tumors in re-transplanted B6 or PD-1−/− hosts with an unlocked PD-1/PD-L1 control system. CD8 T cell-mediated elimination of HBshi/pCCL, together with the attenuation of the negative restraints of HBs in the tumor cells, like ER-stress, reveals a novel mechanism to unleash highly aggressive HBslo/pCCL-ex immune-escape variants. Under certain conditions, HBs-specific CD8 T-cell responses thus potentiate tumor growth, an aspect that should be considered for therapeutic vaccination strategies against chronic HBV infection and liver tumors.</p

Heterologous DNA-prime/protein-boost immunization with a monomeric SARS-CoV-2 spike antigen redundantizes the trimeric receptor-binding domain structure to induce neutralizing antibodies in old mice

A multitude of alterations in the old immune system impair its functional integrity. Closely related, older individuals show, for example, a reduced responsiveness to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines. However, systematic strategies to specifically improve the efficacy of vaccines in the old are missing or limited to simple approaches like increasing the antigen concentration or injection frequencies. We here asked whether the intrinsic, trimeric structure of the SARS-CoV-2 spike (S) antigen and/or a DNA- or protein-based antigen delivery platform affects priming of functional antibody responses particularly in old mice. The used S-antigens were primarily defined by the presence/absence of the membrane-anchoring TM domain and the closely interlinked formation/non-formation of a trimeric structure of the receptor binding domain (S-RBD). Among others, we generated vectors expressing prefusion-stabilized, cell-associated (TM+) trimeric “S2-P” or secreted (TM−) monomeric “S6-PΔTM” antigens. These proteins were produced from vector-transfected HEK-293T cells under mild conditions by Strep-tag purification, revealing that cell-associated but not secreted S proteins tightly bound Hsp73 and Grp78 chaperones. We showed that both, TM-deficient S6-PΔTM and full-length S2-P antigens elicited very similar S-RBD-specific antibody titers and pseudovirus neutralization activities in young (2–3 months) mice through homologous DNA-prime/DNA-boost or protein-prime/protein-boost vaccination. The trimeric S2-P antigen induced high S-RBD-specific antibody responses in old (23-24 months) mice through DNA-prime/DNA-boost vaccination. Unexpectedly, the monomeric S6-PΔTM antigen induced very low S-RBD-specific antibody titers in old mice through homologous DNA-prime/DNA-boost or protein-prime/protein-boost vaccination. However, old mice efficiently elicited an S-RBD-specific antibody response after heterologous DNA-prime/protein-boost immunization with the S6-PΔTM antigen, and antibody titers even reached similar levels and neutralizing activities as in young mice and also cross-reacted with different S-variants of concern. The old immune system thus distinguished between trimeric and monomeric S protein conformations: it remained antigen responsive to the trimeric S2-P antigen, and a simple change in the vaccine delivery regimen was sufficient to unleash its reactivity to the monomeric S6-PΔTM antigen. This clearly shows that both the antigen structure and the delivery platform are crucial to efficiently prime humoral immune responses in old mice and might be relevant for designing “age-adapted” vaccine strategies

Exploring the induction of preproinsulin-specific Foxp3+ CD4+ Treg cells that inhibit CD8+ T cell-mediated autoimmune diabetes by DNA vaccination

DNA vaccination is a promising strategy to induce effector T cells but also regulatory Foxp3(+) CD25(+) CD4(+) Treg cells and inhibit autoimmune disorders such as type 1 diabetes. Little is known about the antigen requirements that facilitate priming of Treg cells but not autoreactive effector CD8(+) T cells. We have shown that the injection of preproinsulin (ppins)-expressing pCI/ppins vector into PD-1- or PD-L1-deficient mice induced K(b)/A12-21-monospecific CD8(+) T cells and autoimmune diabetes. A pCI/ppinsΔA12-21 vector (lacking the critical K(b)/A12-21 epitope) did not induce autoimmune diabetes but elicited a systemic Foxp3(+) CD25(+) Treg cell immunity that suppressed diabetes induction by a subsequent injection of the diabetogenic pCI/ppins. TGF-β expression was significantly enhanced in the Foxp3(+) CD25(+) Treg cell population of vaccinated/ppins-primed mice. Ablation of Treg cells in vaccinated/ppins-primed mice by anti-CD25 antibody treatment abolished the protective effect of the vaccine and enabled diabetes induction by pCI/ppins. Adoptive transfer of Treg cells from vaccinated/ppins-primed mice into PD-L1(-/-) hosts efficiently suppressed diabetes induction by pCI/ppins. We narrowed down the Treg-stimulating domain to a 15-residue ppins76-90 peptide. Vaccine-induced Treg cells thus play a crucial role in the control of de novo primed autoreactive effector CD8(+) T cells in this diabetes model.Published versio

Preproinsulin Designer Antigens Excluded from Endoplasmic Reticulum Suppressed Diabetes Development in NOD Mice by DNA Vaccination

DNA vaccines against autoimmune type 1 diabetes (T1D) contain a nonpredictable risk to induce autoreactive T cell responses rather than a protective immunity. Little is known if (and how) antigen expression and processing requirements favor the induction of autoreactive or protective immune responses by DNA immunization. Here, we analyzed whether structural properties of preproinsulin (ppins) variants and/or subcellular targeting of ppins designer antigens influence the priming of effector CD8+ T cell responses by DNA immunization. Primarily, we used H-2b RIP-B7.1 tg mice, expressing the co-stimulator molecule B7.1 in beta cells, to identify antigens that induce or fail to induce autoreactive ppins-specific (Kb/A12-21 and/or Kb/B22-29) CD8+ T cell responses. Female NOD mice, expressing the diabetes-susceptible H-2g7 haplotype, were used to test ppins variants for their potential to suppress spontaneous diabetes development. We showed that ppins antigens excluded from expression in the endoplasmic reticulum (ER) did not induce CD8+ T cells or autoimmune diabetes in RIP-B7.1 tg mice, but efficiently suppressed spontaneous diabetes development in NOD mice as well as ppins-induced CD8+ T cell-mediated autoimmune diabetes in PD-L1−/− mice. The induction of a ppins-specific therapeutic immunity in mice has practical implications for the design of immune therapies against T1D in individuals expressing different major histocompatibility complex (MHC) I and II molecules. Keywords: type 1 diabetes, mouse models, DNA vaccines, endoplasmic reticulum, preproinsulin/proinsulin antigen

A Missing PD-L1/PD-1 Coinhibition Regulates Diabetes Induction by Preproinsulin-Specific CD8 T-Cells in an Epitope-Specific Manner

<div><p>Coinhibitory PD-1/PD-L1 (B7-H1) interactions provide critical signals for the regulation of autoreactive T-cell responses. We established mouse models, expressing the costimulator molecule B7.1 (CD80) on pancreatic beta cells (RIP-B7.1 tg mice) or are deficient in coinhibitory PD-L1 or PD-1 molecules (PD-L1^−/− and PD-1^−/− mice), to study induction of preproinsulin (ppins)-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD) by DNA-based immunization. RIP-B7.1 tg mice allowed us to identify two CD8 T-cell specificities: pCI/ppins DNA exclusively induced K^b/A_12–21-specific CD8 T-cells and EAD, whereas pCI/ppinsΔA_12–21 DNA (encoding ppins without the COOH-terminal A_12–21 epitope) elicited K^b/B_22–29-specific CD8 T-cells and EAD. Specific expression/processing of mutant ppinsΔA_12–21 (but not ppins) in non-beta cells, targeted by intramuscular DNA-injection, thus facilitated induction of K^b/B_22–29-specific CD8 T-cells. The A_12–21 epitope binds K^b molecules with a very low avidity as compared with B_22–29. Interestingly, immunization of coinhibition-deficient PD-L1^−/− or PD-1^−/− mice with pCI/ppins induced K^b/A_12–21-monospecific CD8 T-cells and EAD but injections with pCI/ppinsΔA_12–21 did neither recruit K^b/B_22–29-specific CD8 T-cells into the pancreatic target tissue nor induce EAD. PpinsΔA_12–21/(K^b/B_22–29)-mediated EAD was efficiently restored in RIP-B7.1⁺/PD-L1^−/− mice, differing from PD-L1^−/− mice only in the tg B7.1 expression in beta cells. Alternatively, an ongoing beta cell destruction and tissue inflammation, initiated by ppins/(K^b/A_12–21)-specific CD8 T-cells in pCI/ppins+pCI/ppinsΔA_12–21 co-immunized PD-L1^−/− mice, facilitated the expansion of ppinsΔA_12–21/(K^b/B_22–29)-specific CD8 T-cells. CD8 T-cells specific for the high-affinity K^b/B_22–29- (but not the low-affinity K^b/A_12–21)-epitope thus require stimulatory ´help from beta cells or inflamed islets to expand in PD-L1-deficient mice. The new PD-1/PD-L1 diabetes models may be valuable tools to study under well controlled experimental conditions distinct hierarchies of autoreactive CD8 T-cell responses, which trigger the initial steps of beta cell destruction or emerge during the pathogenic progression of EAD.</p></div

Priming of K^b/B_22–29-specific CD8 T-cell responses and EAD by mutant ppins antigens.

<p>(<b>A</b>) Map of the expression vectors pCI/ppinsΔA_12–21, pCI/SP-B (encoding the ER-targeting signal peptide and the insulin B-chain) and pCI/SP-B-C (encoding the ER-targeting signal peptide up to the C-peptide). The position of the K^b/B_22–29 epitope (○) is indicated. (<b>B</b>) RIP-B7.1 tg mice were immunized with pCI/ppinsΔA_12–21 (group 1, n = 4), pCI/SP-B (group 2, n = 8) or pCI/SP-B-C DNA (group 3, n = 8) and cumulative diabetes incidences were determined. The statistical significance of diabetes induction in immunized mice was determined using the log-rank test. Values of P<0.05 were considered significant. (<b>C</b>) HEK-293 cells were transiently transfected with pCI (lane 1), pCI/ppins (lane 2) or pCI/ppinsΔA_12–21DNA (lane 3). Cells were labeled with ³⁵S-methionine/cysteine, lysed and immunoprecipitated with an anti-insulin (H86) Ab and protein G sepharose. Immunoprecipitates were processed for SDS-PAGE, followed by fluorography of the gels. The position of pins is indicated (<b>D</b>) HEK-293 cells were transiently transfected with pCI/ppins (lane 1) or pCI/ppinsΔA_12–21 (lanes 2–4). At 28 h after transfection, cells were either non-treated (lanes 1 and 2), or incubated for 6 h with the proteasome-inhibitors expoxymycin (ep; lane 3) or lactacystein (lac; lane 4) and subsequently lysed. Total cell extracts were subjected to high resolution tricine-urea-SDS-PAGE (16%) followed by anti-insulin (H86) specific western blotting.</p

Recruitment of different ‘bystander’ cell populations into the pancreatic target tissue.

<p>PD-L1^−/− mice were immunized with both, pCI/ppins+pCI/ppinsΔA_12–21 vectors into the right and the left tibialis anterior muscles, respectively. Pancreata of representative healthy (at 3 days post immunization) (A) or early diabetic mice (at 15–20 days post immunization) (B) were analyzed histologically for insulin expression (insulin) and influx of CD4⁺ T-cells (CD4⁺), macrophages (F4/80⁺) or DCs (CD11c⁺).</p

Determination of K^b/B_22–29-tetramer⁺ CD8 T-cells in diabetic RIP-B7.1 tg mice.

<p>(<b>A</b>) TAP-deficient RMA-S cells were either not pulsed (−/−) or pulsed for 6 h with high doses (100 µg/ml) of K^b/A_12-N21A or K^b/B_22–29 peptides, followed by surface staining of trimeric K^b-molecules and FCM. (<b>B</b>) RIP-B7.1 tg mice were immunized with pCI, pCI/ppins or pCI/ppinsΔA_12–21. CD8 T-cells were prepared from pancreata of early diabetic (pCI/ppins, pCI/ppinsΔA_12–21) or non-diabetic (pCI) mice and directly stained with K^b/B_22–29-tetramers. Primary FACS data are shown for representative mice. The actual percentage of K^b/B_22–29-tetramer⁺ CD8 T-cells within the pancreas-infiltrating CD8 T-cell population is shown in brackets. (<b>C</b>) The numbers of K^b/B_22–29-tetramer⁺ CD8 T-cells were determined during the course of pCI/ppinsΔA_12–21-mediated EAD: group 1, health mice (n = 3) with blood glucose levels <200 mg/dl; group 2, early diabetic mice (n = 3) with blood glucose levels between 250–350 mg/dl; group 3, diabetic mice (n = 3) with severe diabetes (i.e., blood glucose levels between 400–550 mg/dl). Pancreata of representative mice out of groups 1 to 3 were analyzed histologically for CD8 T-cell influx (CD8+) or stained with hematoxylin-eosin (H&E).</p

The RIP-B7.1 diabetes model.

<p>(<b>A</b>) Map of ppins antigens. The expression vectors encoding the ppins and the mutant ppinsΔA_12–21 are shown. The signal peptide (SP), the insulin B- and A- chains, the C-peptide and the position and sequences of the K^b/A_12–21 epitope (•), its K^b/A_12-N21A variant and of the newly identified K^b/B_22–29 epitope (○) are indicated. (<b>B,C</b>) RIP-B7.1 tg mice were immunized with pCI (groups 1, n = 6), pCI/ppins (groups 2, n = 6) or pCI/ppinsΔA_12–21 (groups 3, n = 6). At indicated times after immunization, blood glucose levels (<b>B</b>) and cumulative diabetes incidences (<b>C</b>) were determined. The statistical significance of diabetes induction in immunized mice was determined using the log-rank test. Values of P<0.05 were considered significant. (<b>D</b>) CD8 T-cells were prepared from pancreata of pCI/ppinsΔA_12–21-immune and diabetic RIP-B7.1 tg mice. Pancreatic cell preparations from ten mice were pooled and restimulated <i>ex vivo</i> for 16 hours with a ppins-specific peptide library (i.e., 10 mers with two amino acids offset) and frequencies of IFNγ⁺ CD8 T-cells were determined by by flow cytometry (FCM). The mean % of IFNγ⁺ CD8 T-cells in the pancreatic CD8 T-cell population (obtained from two independent experiments) are shown. CD8 T-cell frequencies <0.05% are defined negative. (<b>E</b>) RIP-B7.1 tg mice were immunized with pCI (group 1), pCI/ppins (group 2), pCI/ppinsΔA_12–21 (group 3) or pCI/ppins and pCI/ppinsΔA_12–21 (group 4). In group 4, the indicated plasmids were injected into the right and the left tibialis anterior muscles, respectively. CD8 T-cells were prepared from pancreata of diabetic (groups 2–4) or non-diabetic (group 1) mice and restimulated <i>ex vivo</i> with A_12-N21A or B_22–29 peptides. Specific IFNγ⁺ CD8 T-cell frequencies were determined by FCM. The mean % of IFNγ⁺ CD8 T-cells in the pancreatic CD8 T-cell population (±SD) of a representative experiment (n = 3 mice per group) is shown. The statistical significance of differences between A_12-N21A- (groups 2 and 4) and K^b/B_22–29-specific CD8 T-cell frequencies (groups 3 and 4) was determined by the unpaired Student’s t-test (ns, not significant).</p