Experimental vaccination of sheep and cattle against tick infestation using recombinant 5′‐nucleotidase

Limited prior evidence suggests that 5′‐nucleotidase, an ectoenzyme principally located in the Malpighian tubules of the tick Rhipicephalus (Boophilus) microplus, could be an effective antigen in an anti‐tick vaccine. To assess this, recombinant 5′‐nucleotidase was expressed in Escherichia coli and used in vaccination trials with both sheep and cattle. Vaccinated sheep were challenged with freshly moulted adult ticks. Those with high titres of anti‐nucleotidase antibodies showed significant protection against tick infestation, although protection was less than that found with the previously characterized antigen, Bm86. Cattle were vaccinated, in separate groups, with 5′‐nucleotidase, Bm86 and both antigens combined. Cattle, as the natural host, were challenged with larval ticks. Although Bm86 showed typical efficacy, no significant protection was seen in cattle vaccinated with 5′‐nucleotidase. Cattle receiving a dual antigen formulation were no better protected than those receiving Bm86 alone. One possible reason for the difference between host species, namely antibody titre, was examined and shown to be an unlikely explanation. This demonstrates a limitation of using a model host like sheep in vaccine studies.     

Antiplatelet‐derived growth factor (PDGF) activity in the saliva of ixodid ticks is linked with their long mouthparts

The saliva of blood‐feeding arthropods modulates their vertebrate hosts' haemostatic, inflammatory and immune responses to facilitate blood feeding. In a previous study, we showed that salivary gland products from ixodid tick species also manipulate the wound‐healing response by targeting at least four different mammalian growth factors: transforming growth factor β1, hepatocyte growth factor, fibroblast growth factor 2 and platelet‐derived growth factor (PDGF). In addition, species that showed PDGF‐binding activity also inhibited cell proliferation in vitro and induced changes in cell morphology accompanied by disruption of the actin cytoskeleton. Here, we show a correlation between the length of the tick hypostome, the sclerotized feeding tube of the mouthparts inserted into the host's skin and anti‐PDGF activity. This apparent link between hypostome length, and hence the potential depth of skin damage, and PDGF‐binding activity was not apparent for the other growth factors or for other cytokines important in wound healing (keratinocyte growth factor, interleukin 6 and stromal cell‐derived factor 1). However, PDGF‐binding activity was no longer correlated with anti‐cell activities, indicating that an additional as yet unidentified activity in tick saliva may affect cellular changes in wound repair.     

Differential anti-chemokine activity of Amblyomma variegatum adult ticks during blood-feeding

Ticks secrete a cocktail of immunomodulatory molecules in their saliva during blood-feeding, including chemokine-binding factors that help control the activity of host immunocompetent cells. Here we demonstrate differential dynamics of anti IL-8 (CXCL8), MCP-1 (CCL2), MIP-1 (CCL3), RANTES (CCL5) and eotaxin (CCL11) activities in salivary gland extracts of adult Amblyomma variegatum. Unfed male and female ticks showed activity against all the chemokines except CCL5; anti-CCL11 activity was particularly high. However, during feeding the dynamics of anti-chemokine activity differed significantly between males and females, and varied between chemokines. In males, anti-chemokine activities increased, whereas in females they declined or increased slightly as feeding progressed. The exception was anti-CCL11 activity, which declined and then increased in both males and females. Comparison of salivary gland equivalents of individual ticks prepared at various feeding intervals revealed some differences that were most pronounced between individual females fed for 8 days. These observations reflect the feeding behaviour of male and female A. variegatum. They support the concept of 'mate guarding', in which males help their mates to engorge by controlling their host's immune response, and the possibility that ticks benefit from feeding together by exploiting molecular individuality.     

Tick saliva increases production of three chemokines including monocyte chemoattractant protein‐1, a histamine‐releasing cytokine

The effect of Ixodes ricinus tick saliva on the production of various cytokines and chemokines by mouse splenocytes was tested by a cytokine array. We demonstrated a strong upregulation of three chemokines, monocyte chemoattractant protein‐1 (MCP‐1), thymus‐derived chemotactic agent 3 (TCA‐3) and macrophage inflammatory protein 2 (MIP‐2). MCP‐1 could be induced by tick saliva itself. While TCA‐3 and MIP‐2 are engaged in Th2 polarization of the host immune response associated with tick feeding, MCP‐1 may act as a histamine release factor, increasing blood flow into the feeding lesion thus facilitating tick engorgement in the late, rapid feeding phase.     

Ticks infesting wild and domestic animals and humans of Sri Lanka with new host records

An island-wide collection of tick species infesting humans, domesticated and wild animals and questing ticks in domestic and peridomestic environments was carried out during 2009–2011. A total of 30,461 ticks were collected from 30 different hosts and free living stages from the ground. The collection consisted of 22 tick species from 30 different hosts recording 12 tick species from humans, 19 from domesticated animals and 21 from wild animals, with a total of 97 new host records. The most common tick species on humans were Dermacentor auratus and Amblyomma testudinairum, while Haemaphysalis intermedia, Rhipicephalus microplus and Rhipicephalus sanguineus were common in domesticated and wild animals sharing 20 host species. Among the questing ticks, immature D. auratus was the most abundant. Humans and domesticated animals were mostly infested by the nymphal stages while adult ticks were found on wild animals. High number of new host records could be due to domestic animals picking tick species from wildlife and vise versa at the human/animal interface. Habitat destruction due to forest fragmentation has lead to wild animals roaming in urban and semi-urban neighbourhoods increasing the interactions of wild animals with domesticated animals. Wild animals play a significant role as a reservoir of many tick borne infections which can easily be spread to domesticated animals and then to humans via tick infestations. Data in this paper are useful for those interested in tick infesting wild and domestic animals and humans in describing the zoonotic potential of tick borne infections.     

Survey of tick species parasiting domestic ruminants in Ghaemshahr county,Mazandaran province,Iran

ObjectiveTo determine the tick species parasitizing domestic ruminants in Ghaemshahr county in Mazandaran, a Caspian province in the north of Iran.MethodsAbout 361 sheep, 54 goats and 10 cattle of 18 herds in several villages in Ghaemshahr were inspected for tick infestation. Separated ticks were preserved in 70% alcohol and identified.ResultsAbout 323 ticks (207 female, 116 male) were collected, the occurrence of ticks on sheep, goats and cattle were 28.3%, 22.2% and 20.0% respectively. The mean number of ticks on each animal was low (3-5 ticks per animal). Rhipicephalus sanguineus, Rhipicephalus bursa, Ixodes ricinus, Boophilus annulatus, Haemaphysalis punctata and Haemaphysalis numidiana were the tick species we found. Rhipicephalus sanguineus were the most abundant species in the study area. The largest number of ticks were generally present from April to July, mostly in animal ears and tails. Ixodes, Boophilus and Haemaphysalis occurred in mountainous areas of Ghaemshahr, whereas Rhipicephalus were present in both mountains and plains of the study area.ConclusionsThe result of this study is a survey of tick species from domestic animals in Iran and implication of possible prevention measures for diseases transmitted by ticks.     

CXCL13 neutralization reduces the severity of collagen‐induced arthritis

Objective

To investigate the role of CXCL13 in the development and pathogenesis of collagen‐induced arthritis (CIA), and to determine the mechanisms involved in the modulation of arthritogenic response by CXCL13 neutralization.

Methods

Mice were immunized with type II collagen (CII) and treated with anti‐CXCL13 or control antibodies during boosting. Mice were monitored for the development and severity of arthritis. The effects of CXCL13 neutralization on immune response to CII were evaluated by cytokine production by CII‐specific T cells and CII‐specific antibody production. Follicular response in the spleen and in synovial tissue was determined by in situ immunohistology.

Results

Mice receiving neutralizing antibodies to CXCL13 developed significantly less severe arthritis compared with mice injected with phosphate buffered saline or control antibodies. Follicular response both in the spleen and in synovial tissue was inhibited by anti‐CXCL13 treatment. Injection with anti‐CXCL13 antibodies did not significantly affect antigen‐specific recall lymphocyte proliferation or type 1 cytokine production in vitro. Antibody response specific to CII was not inhibited by anti‐CXCL13 treatment. However, anti‐CXCL13 treatment induced significantly higher levels of interleukin‐10 production after in vitro CII stimulation.

Conclusion

Neutralization of CXCL13 inhibits the development of CIA and reduces follicular response in both lymphoid and nonlymphoid tissues. These findings may have important implications regarding the pathogenesis and treatment of autoimmune arthritis.

     

Leishmania tarentolae expressing CXCL‐10 as an efficient immunotherapy approach against Leishmania major‐infected BALB/c mice

Recent findings have demonstrated the suitability of interferon‐gamma‐induced protein 10 (IP‐10) or CXCL‐10 as an immunotherapy tool in treatment of leishmaniasis. This chemokine can overcome Leishmania (L.) infection through inducing nitric oxide (NO) production for parasite elimination. This study was undertaken to investigate the therapeutic effects of recombinant Leishmania tarentolae expressing CXCL‐10 and an expression vector encoding CXCL‐10 (pcDNA‐CXCL‐10‐EGFP) in a model of BALB/c mice susceptible to infection by Leishmania major. The outcome of intervention was examined at 3 weeks post‐treatment by evaluating the parameters of parasite burden (PB), arginase activity, NO and various cytokines such as IFN‐γ, IL‐4, IL‐6 and IL‐10. The results have shown that despite the efficacy of CXCL‐10 expression vector as gene therapy, the live therapy strategy using L. tarentolae expressing CXCL‐10 was more effective in terms of decreasing PB. Nitric oxide production increased, especially in the live therapy approaches. Arginase activity also decreased in all regimens, which demonstrates the potency of the treatment. The overall cytokine production shifted in favour of Th1 responses in the treated mice. Altogether, recombinant L. tarentolae expressing CXCL‐10 represents a promising therapeutic strategy to improve treatment of cutaneous leishmaniasis.     

CXCR2 modulates bone marrow vascular repair and haematopoietic recovery post‐transplant

Murine models of bone marrow transplantation show that pre‐conditioning regimens affect the integrity of the bone marrow endothelium and that the repair of this vascular niche is an essential pre‐requisite for successful haematopoietic stem and progenitor cell engraftment. Little is known about the angiogenic pathways that play a role in the repair of the human bone marrow vascular niche. We therefore established an in vitro humanized model, composed of bone marrow stromal and endothelial cells and have identified several pro‐angiogenic factors, VEGFA, ANGPT1, CXCL8 and CXCL16, produced by the stromal component of this niche. We demonstrate for the first time that addition of CXCL8 or inhibition of its receptor, CXCR2, modulates blood vessel formation in our bone marrow endothelial niche model. Compared to wild type, Cxcr2^?/? mice displayed a reduction in bone marrow cellularity and delayed platelet and leucocyte recovery following myeloablation and bone marrow transplantation. The delay in bone marrow recovery correlated with impaired bone marrow vascular repair. Taken together, our data demonstrate that CXCR2 regulates bone marrow blood vessel repair/regeneration and haematopoietic recovery, and clinically may be a therapeutic target for improving bone marrow transplantation.     

Treatment of rheumatoid arthritis with humanized anti–interleukin‐6 receptor antibody: A multicenter,double‐blind,placebo‐controlled trial

Objective

Interleukin‐6 (IL‐6) is a pleiotropic cytokine that regulates the immune response, inflammation, and hematopoiesis. Overproduction of IL‐6 plays pathologic roles in rheumatoid arthritis (RA), and the blockade of IL‐6 may be therapeutically effective for the disease. This study was undertaken to evaluate the safety and efficacy of a humanized anti–IL‐6 receptor antibody, MRA, in patients with RA.

Methods

In a multicenter, double‐blind, placebo‐controlled trial, 164 patients with refractory RA were randomized to receive either MRA (4 mg/kg body weight or 8 mg/kg body weight) or placebo. MRA was administered intravenously every 4 weeks for a total of 3 months. The clinical responses were measured using the American College of Rheumatology (ACR) criteria.

Results

Treatment with MRA reduced disease activity in a dose‐dependent manner. At 3 months, 78% of patients in the 8‐mg group, 57% in the 4‐mg group, and 11% in the placebo group achieved at least a 20% improvement in disease activity according to the ACR criteria (an ACR20 response) (P < 0.001 for 8‐mg group versus placebo). Forty percent of patients in the 8‐mg group and 1.9% in the placebo group achieved an ACR50 response (P < 0.001). The overall incidences of adverse events were 56%, 59%, and 51% in the placebo, 4‐mg, and 8‐mg groups, respectively, and the adverse events were not dose dependent. A blood cholesterol increase was observed in 44.0% of the patients. Liver function disorders and decreases in white blood cell counts were also observed, but these were mild and transient. There was no increase in antinuclear antibodies or anti‐DNA antibodies. Anti‐MRA antibodies were detected in 2 patients.

Conclusion

Treatment with MRA was generally well tolerated and significantly reduced the disease activity of RA.

     

Effects of pharmacological and genetic disruption of CXCR4 chemokine receptor function in B‐cell acute lymphoblastic leukaemia

B cell acute lymphoblastic leukaemia (B‐ALL) cells express high levels of CXCR4 chemokine receptors for homing and retention within the marrow microenvironment. Bone marrow stromal cells (BMSC) secrete CXCL12, the ligand for CXCR4, and protect B‐ALL cells from cytotoxic drugs. Therefore, the therapeutic use of CXCR4 antagonists has been proposed to disrupt cross talk between B‐ALL cells and the protective stroma. Because CXCR4 antagonists can have activating agonistic function, we compared the genetic and pharmacological deletion of CXCR4 in B‐ALL cells, using CRISPR‐Cas9 gene editing and CXCR4 antagonists that are in clinical use (plerixafor, BKT140). Both genetic and pharmacological CXCR4 inhibition significantly reduced B‐ALL cell migration to CXCL12 gradients and beneath BMSC, and restored drug sensitivity to dexamethasone, vincristine and cyclophosphamide. NOD/SCID/IL‐2rγnull mice injected with CXCR4 gene‐deleted B‐ALL cells had significant delay in disease progression and superior survival when compared to control mice injected with CXCR4 wild‐type B‐ALL cells. These findings indicate that anti‐leukaemia activity of CXCR4 antagonists is primarily due to CXCR4 inhibition, rather than agonistic activity, and corroborate that CXCR4 is an important target to overcome stroma‐mediated drug resistance in B‐ALL.     

Biochemical characteristics of ticks of the families Argasidae and Ixodidae

Polyacrylamide gel electrophoresis was used to study homogenate supernatants from the bloodsucking ticks (Argasidae and Ixodidae) belonging to 8 different genera and species. Differences were found in the electrophoretic mobility of protein fractions in adult ticks of these two different families and between several genera of Ixodes ticks (such as Haemaphysalis, Hyalomma, Dermacentor, Rhipicephalus and Ixodes). Argasidae ticks were found to have generic and even species-specific differences at the nymphal stage of development. A. lahorensis nymph-3 contained 5 protein fractions; O. papillipes had 2 fractions, and O. moubata had 3 fractions. Electrophoretic protein separation showed the equal number of protein fractions within one genus of ticks. At the larval stage, the tick homogenates were always found to have one protein fraction. The hemolymph of O. papillipes and O. moubata ticks displayed differences between the imago and nymph-3. A new biochemical marker was proposed to establish a species affinity.     

Decreased apoptosis of pulmonary PMN in COPD patients with community‐acquired pneumonia

Introduction: Chronic obstructive pulmonary disease (COPD) predisposes for the acquisition of community‐acquired pneumonia (CAP). Objective/Methods: To assess clinically and scientifically suggested disorders in innate immune response during acute phrase and resolution CAP (T2), we evaluated peripheral and pulmonary polymorphnuclear neutrophils (PMN), recovered by induced sputum, from CAP patients with and without COPD with regard to cell activation, interleukin‐8 (CXCL‐8) and CXCL‐8 receptor expression, and apoptosis rate. Results: At T1, COPD patients displayed significantly lower pulmonary PMN apoptosis rates, while total cell count, the amount of macrophages, and vital and necrotic neutrophils in sputum samples were similar between study groups. At T2, there were no differences between study groups or between pulmonary and peripheral compartment. While under systemic steroid treatment apoptosis rates of peripheral and pulmonary PMN at T1 were slightly decreased, there were no significant differences in intrapulmonary CXCL‐8 levels. Regarding cell activation, no significant differences could be seen, neither in comparing study groups nor in pulmonary to peripheral PMN. Conclusion: Elucidating the pathology of suspected disorder in innate immune response, we found decreased apoptosis rates of pulmonary neutrophils in COPD at the peak of CAP indicating an increased inflammatory response, which is independent from anti‐apoptotic cytokines such as CXCL‐8, severity of disease and isolation of bacteria from sputum cultures. Please cite this paper as: Strassburg A, Luers A and Dalhoff K. Decreased apoptosis of pulmonary PMN in COPD patients with community‐acquired pneumonia. The Clinical Respiratory Journal 2010; 4: 111–119.     

Heterogeneity in the effect of different ixodid tick species on human natural killer cell activity

Tick saliva plays a vital role in blood-feeding, including manipulation of the host response to tick infestation. Furthermore, a diverse number of tick-borne pathogens are transmitted to vertebrate hosts via tick saliva, some of which exploit the immunomodulatory activities of their vector's saliva. We report that salivary gland extracts (SGE) derived from Dermacentor reticulatus adult ticks induce a decrease in the natural killer (NK) activity of effector cells obtained from healthy human blood donors. The decrease was observed with SGE from both female and male D. reticulatus fed for either 3 or 5 days on mice, but no significant effect was observed with SGE from unfed ticks or ticks that had fed for 1 day. These results indicate that the tick anti-NK factor(s) is only active after blood-feeding has commenced. Microscopic examination revealed that the first step of NK activity, namely effector/target cell conjugate formation, was affected by SGE. The observed reduction in conjugate formation occurred when effector (but not target) cells were treated with SGE for 30 min, and the effect persisted after 12 h of treatment. Similar but less potent anti-NK activity was detected for SGE from Amblyomma variegatum and Haemaphysalis inermis. By contrast, SGE derived from Ixodes ricinus and Rhipicephalus appendiculatus female ticks did not decrease NK activity. The apparent absence of such activity in these two important vectors of tick-borne viruses suggests that control of NK cells does not play an important role in promoting virus transmission, at least for these particular species.     

Isolation of a Theileria species from Eland (Taurotragus oryx) infective for cattle.

Theileria infections were induced in cattle by feeding ticks on them from 3 sources: (a) adult rhipicephalid ticks obtained from the vegetation in a paddock containing an eland EAO at the Animal Orphanage, Nairobi National Park, Kenya, (b) Rhipicephalus appendiculatus adults fed as nymphs on the same eland, (c) R. pulchellus adults fed as nymphs on an eland W 68 captured in the Machakos district of Kenya. Both eland were harbouring Theileria parasites at the time nymphal ticks were fed. Mild infections were produced when adult ticks from these 3 batches were applied to cattle associated with low numbers of schizonts and piroplasms. The indirect fluorescent antibody test demonstrated that cattle recovered from infections resulting from the above 3 tick batches from eland W 68 and EAO produced antibodies which reacted with schizont antigen of the Theileria species (eland) and Theileria species (Githunguri) which had been isolated from cattle and not to antigens of other Theileria species used. The cattle recovered from the Theileria species (eland) were fully susceptible to a lethal challenge of a T. parva (Muguga) stabilate. It was concluded that the Theileria species (eland) and Theileria species (Githunguri) may be closely related and could represent a new species of Theileria infective to cattle.     

Antibody levels correlate with creatine kinase levels and strength in anti–3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase–associated autoimmune myopathy

Objective

Autoantibodies recognizing 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase (HMGCR) are found in patients with statin‐associated immune‐mediated necrotizing myopathy and, less commonly, in statin‐unexposed patients with autoimmune myopathy. The main objective of this study was to define the association of anti‐HMGCR antibody levels with disease activity.

Methods

Anti‐HMGCR levels, creatine kinase (CK) levels, and strength were assessed in anti‐HMGCR–positive patients. Associations of antibody level with CK level and strength at visit 1 were analyzed in 55 patients, 40 of whom were exposed to statins. In 12 statin‐exposed and 5 statin‐unexposed patients with serum from 5 serial visits, the evolution of antibody levels, CK levels, and strength was investigated.

Results

Antibody levels were associated with CK levels (P < 0.001), arm strength (P < 0.05), and leg strength (P < 0.05) at visit 1, but these associations were only significant among statin‐exposed patients in stratified analyses. With immunosuppressive treatment over 26.2 ± 12.6 months (mean ± SD), antibody levels declined (P < 0.05) and arm abduction strength improved (P < 0.05) in the 17 patients followed up longitudinally. The separate analysis showed that statin‐exposed patients developed decreased antibody levels (P < 0.01), decreased CK levels (P < 0.001), improved arm strength (P < 0.05), and improved hip flexion strength (P < 0.05) with treatment. Anti‐HMGCR antibody levels did not normalize in any patient.

Conclusion

In the entire cohort, initial anti‐HMGCR levels correlated with indicators of disease activity; with immunosuppressive treatment, antibody levels declined and arm strength improved. Statin‐exposed patients had significant improvements in CK levels and strength whereas statin‐unexposed patients did not, suggesting a phenotypic difference between statin‐exposed and statin‐unexposed anti‐HMGCR–positive patients.

     

Oxidative stress and regulation of anti‐oxidant enzymes in cytochrome P4502E1 transgenic mouse model of non‐alcoholic fatty liver

Background and Aim: Reactive oxygen species produced by cytochrome P4502E1 (CYP2E1) are believed to play a role in pathophysiology of non‐alcoholic fatty liver disease (NAFLD). However, little is known about the expression, protein content and activity of anti‐oxidant enzymes and the role of inducible nitric oxide synthase (iNOS), a source of reactive nitrogen species, in NAFLD. In the present study, we evaluate gene expression, protein content and activity of anti‐oxidant enzymes, and iNOS, in a CYP2E1 overexpressing model of non‐alcoholic fatty liver (NAFL). Methods: Non‐transgenic (nTg) and CYP2E1 transgenic (Tg) mice were fed rodent chow for 8 months. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver triglycerides, malondialdehyde and protein carbonyls were measured. Gene expression of NF‐E2‐related factor (Nrf2), superoxide dismutase‐1, ‐2 (SOD‐1,2), catalase (CAT), glutathione peroxidase (GPx), heme oxygenase‐1 (HO‐1) and iNOS were determined. Protein content, activity and nitrosylation of the enzymes were also measured. Results: Tg mice had greater CYP2E1 activity and histological liver injury. MDA and protein carbonyls were increased, indicating insufficient anti‐oxidant response. Gene expression of Nrf2, CAT, GPx, HO‐1 and iNOS were significantly increased. Protein content and enzyme activities of most anti‐oxidant enzymes were not correspondingly increased. iNOS activity and nitrosylation of CAT and SOD was greater in Tg mice liver. Conclusion: Hepatocyte‐specific CYP2E1 overexpression results in increased oxidative stress and nitrosative stress. Several anti‐oxidant enzymes are upregulated. Failure of corresponding increase in total protein and activity of anti‐oxidant enzymes suggests modification/degradation, possibly by nitrosylation, due to increased iNOS activity in a CYP2E1 overexpressing NAFL mouse model.     

Expression of programmed cell death protein 1 and T‐cell immunoglobulin‐ and mucin‐domain‐containing molecule‐3 on peripheral blood CD4+CD8+ double positive T cells in patients with chronic hepatitis C virus infection and in subjects who spontaneously cleared the virus

Chronic hepatitis C virus (HCV) infection is characterized by increased proportion of CD4+CD8+ double positive (DP) T cells, but their role in this infection is unclear. In chronic hepatitis C, immune responses to HCV become functionally exhausted, which manifests itself by increased expression of programmed cell death protein 1 (PD‐1) and T‐cell immunoglobulin‐ and mucin‐domain‐containing molecule‐3 (Tim‐3) on T cells. The aim of our study was to determine PD‐1 and Tim‐3 phenotype of DP T cells in subjects with naturally resolved and chronic HCV infection. Peripheral blood mononuclear cells from 16 patients with chronic infection and 14 subjects who cleared HCV in the past were stained with anti‐CD3, anti‐CD4, anti‐CD8, anti‐PD‐1 and anti‐Tim‐3 antibodies and, in 12 HLA‐A*02‐positive subjects, MHC class I pentamer with HCV NS3₁₄₀₆ epitope. In chronic and past HCV infection, proportions of total DP T cells and PD‐1+ DP T cells were similar but significantly higher than in healthy controls. DP T cells were more likely to be PD‐1+ than either CD4+ or CD8+ single positive (SP) T cells. HCV‐specific cells were present in higher proportions among DP T cells than among CD8+ SP T cells in both patient groups. Furthermore, while the majority of HCV‐specific DP T cells were PD‐1+, the proportion of HCV‐specific CD8+ T cells which were PD‐1+ was 4.9 and 1.9 times lower (chronic and past infection, respectively). PD‐1 and Tim‐3 were predominantly expressed on CD4^highCD8^low and CD4^lowCD8^high cells, respectively, and co‐expression of both markers was uncommon.