Therapeutic cancer vaccines show promise in preclinical studies, yet their clinical efficacy is limited. Increased recruitment of immune cells into tumors and suppression of the immune suppressive ...tumor environment are critical components toward effective cancer immunotherapies. Here, we report how local low‐dose irradiation, alone or with a therapeutic immunization based on Semliki Forest virus (SFV) against human papillomavirus (HPV)‐related cancer, influences these immune mechanisms. We first demonstrated that immunization with SFVeE6,7 or SFVeOVA, replicon particles expressing either HPV16 E6/E7 or ovalbumin, resulted in an antigen‐specific migration of CD8+ T cells into HPV‐ and OVA‐specific tumors. Local low‐dose tumor irradiation alone resulted in a 2‐fold increase of intratumoral CD8+ T cells. When 14 Gy irradiation was combined with immunization, intratumoral numbers of CD8+ T cells increased 10‐fold and the number of CD8+ T cells specific for the E7‐ epitope increased more than 20‐fold. Irradiation alone however also increased the number of intratumoral myeloid‐derived suppressor cells (MDSCs) 3.5‐fold. Importantly, this number did not further increase when combined with immunization. As a result, the ratio of antigen‐specific CD8+ T cells and MDSCs in tumors increased up to 85‐fold compared to the control. We furthermore demonstrated that following irradiation CCR2 and CCL2, CXCR6 and CCL16, chemokines and ligands involved in tumor homing of immune cells, were significantly up regulated. This study demonstrates that local low‐dose tumor irradiation influences the intratumoral immune population induced by SFVeE6,7 immunization by a strong increase in the ratio of antitumoral to immune suppressive cells, thus changing the intratumoral immune balance in favor of antitumor activity.
What's new?
Cancer immunotherapy often fails because of the immunosuppressive environment of the tumor and the lack of infiltration with antigen‐specific T cells. This study demonstrates that low‐dose tumor irradiation combined with a cancer vaccine can change the intratumoral immune balance in favor of antitumor immune activation. Therapeutic immununization of mice with a Semliki Forest virus‐based vector expressing human papilloma virus E6/E7 oncoproteins resulted in an 85‐fold increase in the ratio of antitumoral CD8+ T cells to immune suppressive myeloid‐derived suppressor cells at the tumor site. This raises the hope that a combination regimen might be a promising strategy to enhance the success of therapeutic tumor vaccination.
Tuberculosis (TB) is among the leading causes of morbidity and mortality. The causative agent, Mycobacterium tuberculosis (Mtb), has evolved virulent factors for entry, survival, multiplication and ...immune evasion. Rv2031 (also called alpha crystallin, hspX, 16-kDa antigen), one of the most immunogenic latency antigens, is believed to play a key role in long-term viability of Mtb. Here, we report the dynamics of pro-inflammatory (IFN-γ, TNF-α) and anti-inflammatory (IL-10) cytokines against Rv2031 using whole blood assay in human cohorts in a TB endemic setting. Cytokine responses to ESAT-6-CFP-10 were also measured for comparison. Blood samples were collected from smear positive pulmonary TB patients and their contacts at baseline, 6 and 12 months, and from community controls at entry. At baseline, 54.4% of controls and 73.2% of contacts were QFT-GIT test positive. Baseline IFN-γ, TNF-α and IL-10 responses to Rv2031 were significantly higher in controls compared to contacts and untreated patients (p<0.001). Furthermore, untreated patients had significantly higher TNF-α and IL-10 responses to Rv2031 compared to contacts (p<0.001). In contacts and treated patients, IFN-γ, TNF-α and IL-10 responses to Rv2031 significantly increased over 12 months (p<0.0001) and became comparable with the corresponding levels in controls. There was a positive and significant correlation between Rv2031 and ESAT-6-CFP-10 specific cytokine responses in each study group. The fact that the levels of IFN-γ, TNF-α and IL-10 against Rv2031 were highest during latent TB infection may indicate their potential as markers of protection against TB. Taken together, the findings of this study suggest the potential of IFN-γ, TNF-α and IL-10 against Rv2031 as biomarkers of the host response to Mtb during convalescence from, and the absence of, active tuberculosis.
Antibiotic resistance poses rapidly increasing global problems in combatting multidrug-resistant (MDR) infectious diseases like MDR tuberculosis, prompting for novel approaches including ...host-directed therapies (HDT). Intracellular pathogens like Salmonellae and Mycobacterium tuberculosis (Mtb) exploit host pathways to survive. Only very few HDT compounds targeting host pathways are currently known. In a library of pharmacologically active compounds (LOPAC)-based drug-repurposing screen, we identify multiple compounds, which target receptor tyrosine kinases (RTKs) and inhibit intracellular Mtb and Salmonellae more potently than currently known HDT compounds. By developing a data-driven in silico model based on confirmed targets from public databases, we successfully predict additional efficacious HDT compounds. These compounds target host RTK signaling and inhibit intracellular (MDR) Mtb. A complementary human kinome siRNA screen independently confirms the role of RTK signaling and kinases (BLK, ABL1, and NTRK1) in host control of Mtb. These approaches validate RTK signaling as a drugable host pathway for HDT against intracellular bacteria.
Abstract
Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic ...peptide vaccine elicits an accruing CD8
+
T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8
+
T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8
+
effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8
+
T cell response leading to protection against otherwise lethal SARS-CoV-2 infection.
Tuberculosis (TB) remains one of the most deadly infectious diseases. One-third to one-fourth of the human population is estimated to be infected with Mycobacterium tuberculosis (Mtb) without showing ...clinical symptoms, a condition called latent TB infection (LTBI). Diagnosis of Mtb infection is based on the immune response to a mixture of mycobacterial antigens (PPD) or to Mtb specific ESAT-6/CFP10 antigens (IGRA), highly expressed during the initial phase of infection. However, the immune response to PPD and IGRA antigens has a low power to discriminate between LTBI and PTB. The T-cell response to a group of so-called latency (DosR-regulon-encoded) and Resuscitation Promoting (Rpf) antigens of Mtb has been proved to be significantly higher in LTBI compared to active TB across many populations, suggesting their potential use as biomarkers to differentiate latent from active TB.
PBMCs from a group LTBI (n = 20) and pulmonary TB patients (PTB, n = 21) from an endemic community for TB of the city of Medellín, Colombia, were in vitro stimulated for 7 days with DosR- (Rv1737c, Rv2029c, and Rv2628), Rpf- (Rv0867c and Rv2389c), the recombinant fusion protein ESAT-6-CFP10 (E6-C10)-, or PPD-antigen. The induced IFNγ levels detectable in the supernatants of the antigen-stimulated cells were then used to calculate specificity and sensitivity in discriminating LTBI from PTB, using different statistical approaches.
IFNγ production in response to DosR and Rpf antigens was significantly higher in LTBI compared to PTB. ROC curve analyses of IFNγ production allowed differentiation of LTBI from PTB with areas under the curve higher than 0.70. Furthermore, Multiple Correspondence Analysis (MCA) revealed that LTBI is associated with higher levels of IFNγ in response to the different antigens compared to PTB. Analysis based on decision trees showed that the IFNγ levels produced in response to Rv2029c was the leading variable that best-classified disease status. Finally, logistic regression analysis predicted that IFNγ produced by PBMCs in response to E6-C10, Rv2029c, Rv0867c (RpfA) and Rv2389c (RpfA) antigens correlates best with the probability of being latently infected.
The Mtb antigens E6-C10, Rv2029c (PfkB), Rv0867c (RpfA) and Rv2389c (RpfA), may be potential candidates to discriminate LTBI from PTB.
T-cell responses against dormancy-, resuscitation-, and reactivation-associated antigens of Mycobacterium tuberculosis are candidate biomarkers of latent infection in humans.
We established an assay ...based on two rounds of in vitro restimulation and intracellular cytokine analysis that detects T-cell responses to antigens expressed during latent M. tuberculosis infection. Comparison between active pulmonary tuberculosis (TB) patients and healthy latently M. tuberculosis-infected donors (LTBI) revealed significantly higher T-cell responses against 7 of 35 tested M. tuberculosis latency-associated antigens in LTBI. Notably, T cells specific for Rv3407 were exclusively detected in LTBI but not in TB patients. The T-cell IFNgamma response against Rv3407 in individual donors was the most influential factor in discrimination analysis that classified TB patients and LTBI with 83% accuracy using cross-validation. Rv3407 peptide pool stimulations revealed distinct candidate epitopes in four LTBI.
Our findings further support the hypothesis that the latency-associated antigens can be exploited as biomarkers for LTBI.
One half of a group of 20 patients with human papillomavirus type 16 (HPV16)-induced vulvar intraepithelial neoplasia grade 3 displayed a complete regression (CR) after therapeutic vaccination with ...HPV16 E6/E7 synthetic long peptides. Patients with relatively larger lesions generally did not display a CR. To investigate immune correlates of treatment failure, patients were grouped according to median lesion size at study entry, and HPV16-specific immunity was analyzed at different time points by complementary immunological assays. The group of patients with smaller lesions displayed stronger and broader vaccine-prompted HPV16-specific proliferative responses with higher IFNγ (P = 0.0003) and IL-5 (P < 0.0001) levels than patients with large lesions. Characteristically, this response was accompanied by a distinct peak in cytokine levels after the first vaccination. In contrast, the patient group with larger lesions mounted higher frequencies of HPV16-specific CD4⁺CD25⁺Foxp3⁺ T cells (P = 0.005) and displayed a lower HPV16-specific IFNγ/IL-10 ratio after vaccination (P < 0.01). No disparity in T memory immunity to control antigens was found, indicating that the differences in HPV-specific immunity did not reflect general immune failure. We observed a strong correlation between a defined set of vaccine-prompted specific immune responses and the clinical efficacy of therapeutic vaccination. Notably, a high ratio of HPV16-specific vaccine-prompted effector T cells to HPV16-specific CD4⁺CD25⁺Foxp3⁺ T cells was predictive of clinical success. Foxp3⁺ T cells have been associated previously with impaired immunity in malignancies. Here we demonstrate that the vaccine-prompted level of this population is associated with early treatment failure.
Hypoxia and low concentrations of nitric oxide have been reported to upregulate in vitro gene expression of 48 proteins of the dormancy (DosR) regulon of Mycobacterium tuberculosis. These proteins ...are thought to be essential for the survival of bacteria during persistence in vivo and are targeted by the immune system during latent infection in humans. Here we have analyzed the immunogenicity of eight DosR regulon-encoded antigens by plasmid DNA vaccination of BALB/c and C57BL/6 mice, i.e., Rv1733c, Rv1738, Rv2029c (pfkB), Rv2031c/hspX (acr), Rv2032 (acg), Rv2626c, Rv2627c, and Rv2628. Strong humoral and/or cellular Th1-type (interleukin-2 and gamma interferon) immune responses could be induced against all but one (Rv1738) of these antigens. The strongest Th1 responses were measured following vaccination with DNA encoding Rv2031c and Rv2626c. Using synthetic 20-mer overlapping peptides, 11 immunodominant, predicted major histocompatibility complex class II-restricted epitopes and one Kd-restricted T-cell epitope could be identified. BALB/c and (B6D2)F₁ mice persistently infected with M. tuberculosis developed immune responses against Rv1733c, Rv2031c, and Rv2626c. These findings have implications for proof-of-concept studies in mice mimicking tuberculosis (TB) latency models and their extrapolation to humans for potential new vaccination strategies against TB.
High serum levels of interleukin‐6 (IL‐6) correlate with poor prognosis and chemotherapy resistance in several cancers. The underlying mechanisms and its effects on immunotherapy are largely unknown. ...To address this, we developed a human papillomavirus type 16 (HPV16)‐associated tumor model expressing IL‐6 to investigate the impact of tumor‐expressed IL‐6 during cisplatin chemotherapy and HPV16 synthetic long peptide vaccination as immunotherapy. The effects of tumor‐produced IL‐6 on tumor growth, survival and the tumor microenvironment were analyzed. Our data demonstrated that tumor‐produced IL‐6 conferred resistance to cisplatin and therapeutic vaccination. This was not caused by a changed in vitro or in vivo growth rate of tumor cells, or a changed sensitivity of tumor cells to chemotherapy or T‐cell‐mediated killing. Furthermore, no overt differences in the frequencies of tumor‐infiltrating subsets of T cells or CD11b+ myeloid cells were observed. IL‐6, however, affected the systemic and local function of myeloid cells, reflected by a strong reduction of major histocompatibility complex (MHC) class II expression on all major myeloid cell subtypes. Resistance to both therapies was associated with a changed intratumoral influx of MHC class II+ myeloid cells toward myeloid cells with no or lower MHC class II expression. Importantly, while these IL‐6‐mediated effects provided resistance to the immunotherapy and chemotherapy as single therapies, their combination still successfully mediated tumor control. In conclusion, IL‐6‐mediated therapy resistance is caused by an extrinsic mechanism involving an impaired function of intratumoral myeloid cells. The fact that resistance can be overcome by combination therapies provides direction to more effective therapies for cancer.
What's new?
Interleukin‐6 (IL‐6) cytokine has multiple effects on hematopoiesis and immune function and typically circulates at low levels. In cancer, however, IL‐6 serum levels are significantly elevated, with suspected impacts on tumor behavior. In this study, using a mouse model of human papillomavirus‐induced cancer with IL‐6 expression, the authors show that tumor‐produced IL‐6 confers resistance to both chemotherapy and immunotherapy. Resistance was associated with impaired myeloid cell maturation, with no evidence of involvement of mechanisms intrinsic to tumor cells. Resistance was overcome by combining chemotherapy and immunotherapy, providing insight into a potentially effective therapeutic approach for cancers with IL‐6‐mediated resistance.
HLA‐E presented antigens are interesting targets for vaccination given HLA‐Es’ essentially monomorphic nature. We have shown previously that Mycobacterium tuberculosis (Mtb) peptides are presented by ...HLA‐E to CD8+ effector T cells, but the precise phenotype and functional capacity of these cells remains poorly characterized. We have developed and utilized in this study a new protocol combining HLA‐E tetramer with intracellular staining for cytokines, transcription factors and cytotoxic molecules to characterize these cells in depth. We confirm in this study the significantly increased ex vivo frequency of Mtb‐peptide/HLA‐E‐TM+ CD8+ T cells in the circulation of patients with active tuberculosis (TB). HLA‐E restricted CD8+ T cells from TB patients produced more IL‐13 than cells from controls or subjects with latent tuberculosis infection (LTBI). Compared to total CD8+ T cells, HLA‐E restricted cells produced more IFNγ, IL‐4, IL‐10, and granulysin but less granzyme‐A. Moreover, compared to “classical” Mtb specific HLA‐A2 restricted CD8+ T cells, HLA‐E restricted CD8+ T cells produced less TNFα and perforin, but more IL‐4. In conclusion, HLA‐E restricted‐ Mtb specific cells can produce Th2 cytokines directly.
The combination of tetramer staining with intracellular cytokine staining allowed the detailed characterization of T cells recognizing Mtb in the context of HLA‐E. Here we demonstrate that HLA‐E restricted, Mtb‐specific CD8+ T cells produce Th2 cytokines IL‐4 and IL‐13 directly and have a cytokine profile different from HLA‐A2 restricted Mtb specific T cells.
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