Activation and route of administration both determine the ability of bone marrow-derived dendritic cells to accumulate in secondary lymphoid organs and prime CD8<Superscript>+</Superscript> T cells against tumors

Aims

To examine the effects of route of administration and activation status on the ability of dendritic cells (DC) to accumulate in secondary lymphoid organs, and induce expansion of CD8⁺ T cells and anti-tumor activity.

Methods

DC from bone marrow (BM) cultures were labeled with fluorochromes and injected s.c. or i.v. into naïve mice to monitor their survival and accumulation in vivo. Percentages of specific CD8⁺ T cells in blood and delayed tumor growth were used as readouts of the immune response induced by DC immunization.

Results

The route of DC administration was critical in determining the site of DC accumulation and time of DC persistence in vivo. DC injected s.c. accumulated in the draining lymph node, and DC injected i.v. in the spleen. DC appeared in the lymph node by 24 h after s.c. injection, their numbers peaked at 48 h and declined at 96 h. DC that had spontaneously matured in vitro were better able to migrate compared to immature DC. DC were found in the spleen at 3 h and 24 h after i.v. injection, but their numbers were low and declined by 48 h. Depending on the tumor cell line used, DC injected s.c. were as effective or more effective than DC injected i.v. at inducing anti-tumor responses. Pre-treatment with LPS increased DC accumulation in lymph nodes, but had no detectable effect on accumulation in the spleen. Pre-treatment with LPS also improved the ability of DC to induce CD8⁺ T cell expansion and anti-tumor responses, regardless of the route of DC administration.

Conclusions

Injection route and activation by LPS independently determine the ability of DC to activate tumor-specific CD8⁺ T cells in vivo.

     

Impact of ageing on the response and repertoire of influenza virus-specific CD4 T cells

Background

Ageing has been shown to reduce CD8 T cell repertoire diversity and immune responses against influenza virus infection in mice. In contrast, less is known about the impact of ageing on CD4 T cell repertoire diversity and immune response to influenza virus infection.

Results

The CD4 T cell response was followed after infection of young and aged C57BL/6 mice with influenza virus using a tetramer specific for an immunodominant MHC class II epitope of the influenza virus nucleoprotein. The appearance of virus-specific CD4 T cells in the lung airways of aged mice was delayed compared to young mice, but the overall peak number and cytokine secretion profile of responding CD4 T cells was not greatly perturbed. In addition, the T cell repertoire of responding cells, determined using T cell receptor Vβ analysis, failed to show the profound effect of age we previously described for CD8 T cells. The reduced impact of age on influenza-specific CD4 T cells was consistent with a reduced effect of age on the overall CD4 compared with the CD8 T cell repertoire in specific pathogen free mice. Aged mice that were thymectomized as young adults showed an enhanced loss of the epitope-specific CD4 T cell response after influenza virus infection compared with age-matched sham-thymectomized mice, suggesting that a reduced repertoire can contribute to impaired responsiveness.

Conclusions

The diversity of the CD4 T cell repertoire and response to influenza virus is not as profoundly impaired by ageing in C57BL/6 mice as previously shown for CD8 T cells. However, adult thymectomy enhanced the impact of ageing on the response. Understanding the impact of ageing on CD4 T cell responses to influenza virus infection is an important prerequisite for developing better vaccines for the elderly.

     

Early dysregulation of the memory CD8<Superscript>+</Superscript> T cell repertoire leads to compromised immune responses to secondary viral infection in the aged

Background

Virus-specific memory CD8⁺ T cells persist long after infection is resolved and are important for mediating recall responses to secondary infection. Although the number of memory T cells remains relatively constant over time, little is known about the overall stability of the memory T cell pool, particularly with respect to T cell clonal diversity. In this study we developed a novel assay to measure the composition of the memory T cell pool in large cohorts of mice over time following respiratory virus infection.

Results

We find that the clonal composition of the virus-specific memory CD8⁺ T cell pool begins to change within months of the initial infection. These early clonal perturbations eventually result in large clonal expansions that have been associated with ageing.

Conclusions

Maintenance of clonal diversity is important for effective long-term memory responses and dysregulation of the memory response begins early after infection.

     

Comprehensive circular RNA profiling reveals that circular RNA100783 is involved in chronic CD28-associated CD8(+)T cell ageing

Background

Ageing brings about the gradual deterioration of the immune system, also known as immunosenescence. The role of non-coding circular RNA in immunosenescence is under studied. Using circular RNA microarray data, we assembled Comparison groups (C1, C2, C3 and C4) that allowed us to compare the circular RNA expression profiles between CD28(+)CD8(+) T cells and CD28(-)CD8(+) T cells isolated from healthy elderly or adult control subjects. Using a step-wise biomathematical strategy, the differentially-expressed circRNAs were identified in C1 (CD28(+)CD8(+) vs CD28(-)CD8(+)T cells in the elderly) and C4 (CD28(-)CD8(+)T cells in the elderly vs in the adult), and the commonly-expressed circRNA species from these profiles were optimized as immunosenescence biomarkers.

Results

Four overlapping upregulated circular RNAs (100550, 100783, 101328 and 102592) expressed in cross-comparison between C1 and C4 were validated using quantitative polymerase chain reaction. Of these, only circular RNA100783 exhibited significant validation. None of the down-regulated circular RNAs were expressed in the C1 and the C4 cross-comparisons. Therefore, we further predicted circular RNA100783-targeted miRNA-gene interactions using online DAVID annotation. The analysis revealed that a circular RNA100783-targeted miRNA-mRNA network may be involved in alternative splicing, the production of splice variants, and in the regulation of phosphoprotein expression. Considering the hypothesis of splicing-related biogenesis of circRNAs, we propose that circular RNA100783 may play a role in phosphoprotein-associated functions duringCD28-related CD8(+) T cell ageing.

Conclusions

This study is the first to employ circular RNA profiling to investigate circular RNA-micro RNA interactions in ageing human CD8(+)T cell populations and the accompanying loss of CD28 expression. The overlapping expression of circular RNA100783 may represent a novel biomarker for the longitudinal tracking ofCD28-related CD8(+) T cell ageing and global immunosenescence.

     

Age-related differences in polyfunctional T cell responses

Background

A reduced number of naïve T cells along with an accumulation of differentiated cell types in aging have been described but little is known about the polyfunctionality of the T cell responses. In this study we compared the individual and polyfunctional expression of IFN-γ, MIP-1α, TNF-α, perforin, and IL-2 by T cell subsets, including the newly described stem cell like memory T cells (T_SCM), in response to stimulation with superantigen staphylococcal enterotoxin B (SEB) in older (median age 80, n?=?23) versus younger (median age 27; n?=?23) adults.

Results

Older age was associated with a markedly lower frequency of CD8+ naïve T cells (11% vs. 47%; p?<?0.0001) and an expansion in memory T cell subsets including central memory (p?<?0.05), effector memory and effector T cells (p?<?0.001 for both). There was also a decline in CD4+ naïve T cells in older subjects (33% vs. 45%; p?=?0.02). There were no differences in frequencies or polyfunctional profiles of T_SCM between groups. CD8+ naïve cells in the older group had increased expression of all functional parameters measured compared to the younger subjects and exhibited greater polyfunctionality (p?=?0.04). CD4+ naïve T cells in the older group also showed greater polyfunctionality with a TNF-α and IL-2 predominance (p?=?0.005). CD8+ effector memory and effector T cells exhibited increased polyfunctionality in the older group compared with younger (p?=?0.01 and p?=?0.003).

Conclusions

These data suggest that aging does not have a negative effect on polyfunctionality and therefore this is likely not a major contributor to the immunesenescence described with aging.

     

Impaired specific CD8 T cell response with aging is not due to decreased expression of CD90 on TCR transgenic T cells

Background

CD90 (Thy-1) is a small glycoprotein that is particularly abundant on the surface of mouse thymocytes and peripheral T cells, and is often used as a marker in adoptive transfer experiments to distinguish donor and recipient T cells with different CD90 subtypes. We have performed adoptive transfer experiments with T cell receptor transgenic (TCR Tg) mice to study the impaired CD8 T cell response with aging.

Findings

After stimulation with a CD8 T cell epitope, HA_518-524, the response of TCR Tg CD8 T cells from aged mice was decreased compared to the response of TCR Tg T cells from young mice. CD90 expression was also substantially decreased on the TCR Tg CD8 T cells of aged mice. However, the responses of CD90^hi and CD90^low CD8 T cells of the aged mice were similar in both early activation and proliferation, demonstrating that the impaired Tg T cell response with aging is not associated with the altered CD90 expression on CD8 T cells.

Conclusions

The impaired Tg CD8 T cell response in aged mice is not due to age-associated changes in CD90 expression on Tg CD8 T cells.

     

Antibody-dependent CD56+ T cell responses are functionally impaired in long-term HIV-1 infection

Background

Antibody-dependent cellular cytotoxicity (ADCC), which mainly mediated by natural killer (NK) cells, may play a critical role in slowing human immunodeficiency virus type-1 (HIV-1) disease progression and protecting from HIV-1 infection. Besides classic NK cells, CD56+ T cells also have some NK cell-like properties, such as the large granular lymphocyte morphology and the capacity to destroy NK-sensitive target cells. However, little is known about the potentials of antibody-dependent CD56+ T cell responses and the association between antibody-dependent CD56+ T cell responses and HIV-1 disease progression.

Results

In the present study, we showed evidences that, in addition to NK cells, CD56+ T cells could generate degranulation upon CD16 cross-linking. Ex vivo study showed that FcγRIII (CD16)-mediated CD56+ T cell responses were distinctly induced by IgG antibody-bound P815 cells. Comparatively, CD56? T cells and invariant NKT (CD3+ 6B11+) failed to induce antibody-dependent activation. Antibody-dependent CD56+ T cell responses were mainly ascribed to CD4/CD8 double negative subset and were functionally impaired in long-term HIV-1-infected former plasma donors, regardless of hepatitis C virus (HCV) coinfection status. Also, CD56+ T cell-mediated HIV-1-specific antibody-dependent responses were declined in men who have sex with men with HIV-1 infection over 3 years. Finally, we showed that matrix metalloprotease (MMP) inhibitor GM6001 could partially restored antibody-dependent CD56+ T cell responses of chronic HIV-1-infected subjects.

Conclusions

Our results suggested that CD56+ T cells could mediate ADCC responses and the responses were impaired in chronic HIV-1 infection.

     

Adoptive transfer of T<Subscript>reg</Subscript> depleted autologous T cells in advanced renal cell carcinoma

Purpose

CD4⁺CD25⁺ regulatory T (T_reg) cells are present in increased numbers in patients with advanced cancer and CD25⁺ T cell depletion potentiates tumour immunity in animal models. The aim of this study was to assess the feasibility and safety of adoptive transfer of CD25⁺ depleted autologous T cells in patients with advanced renal cell carcinoma and to examine resulting changes in lymphocyte subsets.

Patients and methods

Six patients with advanced renal cell carcinoma underwent leukapheresis followed by conditioning chemotherapy with cyclophosphamide and fludarabine. The autologous leukapheresis product was depleted of CD25⁺ cells using CliniMACS^® System then re-infused into the patient.

Results

Efficient CD25⁺ depletion from all leukapheresis products was achieved and 0.55–5.87 × 10⁷/kg CD3⁺ cells were re-infused. Chemotherapy related haematological toxicity was observed, but blood counts recovered in all patients allowing discharge after a mean inpatient stay of 21 days. One patient subsequently developed a rapidly progressive neurological syndrome. A transient reduction in CD25⁺ subset was noted in the peripheral blood of 5 out of 6 patients with evidence of increased T cell responses to PHA in 4 out of 6 patients. One patient showed increased specific proliferative responses to the tumour associated antigen h5T4 coinciding with the nadir of T_reg cells.

Conclusions

Given the transient nature of the reduction in CD25⁺ subset and the observed toxicity there is a need to explore further strategies to improve the safety and efficacy of this approach. Nevertheless, the results provide proof of concept in potentiation of tumour antigen T cell responses when T_reg cell levels are depleted.

     

Complement C4 induces regulatory T cells differentiation through dendritic cell in systemic lupus erythematosus

Background

Systemic lupus erythematosus (SLE) is a prototypic systemic autoimmune disease. Complement component 4 (C4) has be proved to play a role in pathogenesis of SLE. In the present study, we investigated the effect of C4 on T cells differentiation.

Methods

Thirty SLE patients were included in this study. CD4+ T cells were isolated from healthy subjects, and dendritic cells (DCs) were isolated from healthy subjects or SLE patients. C4 was supplemented to co-incubate with T cells and DCs.

Results

Serum C4 concentration was positively correlated with regulatory T cell (Treg) percentage (R² = 0.5907, p < 0.001) and TGFβ concentration (R² = 0.5641, p < 0.001) in SLE patients. Different concentrations of C4 had no effect on T cells differentiation. Co-incubated T cells with DCs and C4 for 7 days, the Treg percentage and TGF-β concentration were significantly elevated. In addition, pre-treated DCs (from healthy subjects or SLE patients) with C4 and then co-incubated with T cells, the increases of Treg percentage and TGF-β concentration were also observed.

Conclusion

C4 takes part in T cells differentiation to Treg cells via DCs.

     

Cellular responses to <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> erythrocyte membrane protein-1: use of relatively conserved synthetic peptide pools to determine CD4 T cell responses in malaria-exposed individuals in Benin,West Africa

Background

Plasmodium falciparum erythrocyte membrane protein-1, a variant antigen of the malaria parasite, is potentially a target for the immune response. It would be important to determine whether there are CD4 T cells that recognise conserved regions. However, within the relatively conserved region, there is variation. It is not possible to test T cell responses from small field samples with all possible peptides.

Methods

We have aligned sequences that are relatively conserved between several PfEMP1 molecules, and chosen a representative sequence similar to most of the PfEMP1 variants. Using these peptides as pools representing CIDRα, CIDRβ and DBLβ-δ domains, DBLα domain, and EXON 2 domain of PfEMP1, we measured the CD4 T cell responses of malaria-exposed donors from Benin, West Africa by a FACS based assay.

Results

All the three peptide pools elicited a CD4 T cell response in a proportion of malaria-exposed and non-exposed donors. CD4 T cell proliferation occurs at a relatively higher magnitude to peptide pools from the DBLα and EXON 2 in the malaria-exposed donors living in Benin than in the UK malaria-unexposed donors.

Conclusions

These findings suggest that an immunological recall response to conserved peptides of a variant antigen can be measured. Further testing of individual peptides in a positive pool will allow us to determine those conserved sequences recognised by many individuals. These types of assays may provide information on conserved peptides of PfEMP1 which could be useful for stimulating T cells to provide help to P. falciparum specific B cells.

     

Anti-CCR7 therapy exerts a potent anti-tumor activity in a xenograft model of human mantle cell lymphoma

Background

The chemokine receptor CCR7 mediates lymphoid dissemination of many cancers, including lymphomas and epithelial carcinomas, thus representing an attractive therapeutic target. Previous results have highlighted the potential of the anti-CCR7 monoclonal antibodies to inhibit migration in transwell assays. The present study aimed to evaluate the in vivo therapeutic efficacy of an anti-CCR7 antibody in a xenografted human mantle cell lymphoma model.

Methods

NOD/SCID mice were either subcutaneously or intravenously inoculated with Granta-519 cells, a human cell line derived from a leukemic mantle cell lymphoma. The anti-CCR7 mAb treatment (3 × 200 μg) was started on day 2 or 7 to target lymphoma cells in either a peri-implantation or a post-implantation stage, respectively.

Results

The anti-CCR7 therapy significantly delayed the tumor appearance and also reduced the volumes of tumors in the subcutaneous model. Moreover, an increased number of apoptotic tumor cells was detected in mice treated with the anti-CCR7 mAb compared to the untreated animals. In addition, significantly reduced number of Granta-519 cells migrated from subcutaneous tumors to distant lymphoid organs, such as bone marrow and spleen in the anti-CCR7 treated mice. In the intravenous models, the anti-CCR7 mAb drastically increased survival of the mice. Accordingly, dissemination and infiltration of tumor cells in lymphoid and non-lymphoid organs, including lungs and central nervous system, was almost abrogated.

Conclusions

The anti-CCR7 mAb exerts a potent anti-tumor activity and might represent an interesting therapeutic alternative to conventional therapies.

     

pERK-dependent defective TCR-mediated activation of CD4<Superscript>+</Superscript> T cells in end-stage renal disease patients

Background

Patients with end-stage renal disease (ESRD) have an impaired immune response with a prematurely aged T-cell system. Mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK) and p38, regulate diverse cellular programs by transferring extracellular signals into an intracellular response. T cell receptor (TCR)-induced phosphorylation of ERK (pERK) may show an age-associated decline, which can be reversed by inhibiting dual specific phosphatase (DUSP) 6, a cytoplasmic phosphatase with substrate specificity to dephosphorylate pERK. The aim of this study was to assess whether ESRD affects TCR-mediated signaling and explore possibilities for intervening in ESRD-associated defective T-cell mediated immunity.

Results

An age-associated decline in TCR-induced pERK-levels was observed in the different CD4⁺ (P < 0.05), but not CD8⁺, T-cell subsets from healthy individuals (HI). Interestingly, pERK-levels of CD4⁺ T-cell subsets from young ESRD patients were in between young and elderly HI. A differentiation-associated decline in TCR-induced ERK and p38 phosphorylation was observed in T cells, although TCR-induced p38 phosphorylation was not significantly affected by age and/or ESRD. Frequencies of TCR-induced CD69-expressing CD4⁺ T cells declined with age and were positively associated with pERK. In addition, an age-associated tendency of increased expression of DUSP6 was observed in CD4⁺ T cells of HI and DUSP6 expression in young ESRD patients was similar to old HI. Inhibition of DUSP6 significantly increased TCR-induced pERK-levels of CD4⁺ T cells in young and elderly ESRD patients, and elderly HI.

Conclusions

TCR-mediated phosphorylation of ERK is affected in young ESRD patients consistent with the concept of premature immunological T cell ageing. Inhibition of DUSP6 specific for pERK might be a potential intervention enhancing T-cell mediated immunity in ESRD patients.

     

A comprehensive characterization of aggravated aging-related changes in T lymphocytes and monocytes in end-stage renal disease: the iESRD study

Background

Patients with end-stage renal disease (ESRD) exhibit a premature aging phenotype of the immune system. Nevertheless, the etiology and impact of these changes in ESRD patients remain unknown.

Results

Compared to healthy individuals, ESRD patients exhibit accelerated immunosenescence in both T cell and monocyte compartments, characterized by a dramatic reduction in naïve CD4+ and CD8+ T cell numbers but increase in CD8+ T_EMRA cell and proinflammatory monocyte numbers. Notably, within ESRD patients, aging-related immune changes positively correlated not only with increasing age but also with longer dialysis vintage. In multivariable-adjusted logistic regression models, the combination of high terminally differentiated CD8+ T cell level and high intermediate monocyte level, as a composite predictive immunophenotype, was independently associated with prevalent coronary artery disease as well as cardiovascular disease, after adjustment for age, sex, systemic inflammation and presence of diabetes. Levels of terminally differentiated CD8+ T cells also positively correlated with the level of uremic toxin p-cresyl sulfate.

Conclusions

Aging-associated adaptive and innate immune changes are aggravated in ESRD and are associated with cardiovascular diseases. For the first time, our study demonstrates the potential link between immunosenescence in ESRD and duration of exposure to the uremic milieu.

     

Telomere length dynamics in human memory T cells specific for viruses causing acute or latent infections

Background

Declining telomere length (TL) is associated with T cell senescence. While TL in naïve and memory T cells declines with increasing age, there is limited data on TL dynamics in virus-specific memory CD4⁺ T cells in healthy adults. We combined BrdU-labeling of virus-stimulated T cells followed with flow cytometry-fluorescent in situ hybridization for TL determination. We analyzed TL in T cells specific for several virus infections: non-recurring acute (vaccinia virus, VACV), recurring-acute (influenza A virus, IAV), and reactivating viruses (varicella-zoster virus, VZV, and cytomegalovirus, CMV) in 10 healthy subjects. Additionally, five subjects provided multiple blood samples separated by up to 10 years.

Results

VACV- and CMV-specific T cells had longer average TL than IAV-specific CD4⁺ T cells. Although most virus-specific cells were CD45RA^-, we observed a minor population of BrdU+ CD45RA⁺ T cells characterized by long telomeres. Longitudinal analysis demonstrated a slow decline in average TL in virus-specific T cells. However, in one subject, VZV reactivation led to an increase in average TL in VZV-specific memory T cells, suggesting a conversion of longer TL cells from the naïve T cell repertoire.

Conclusions

TLs in memory CD4⁺ T cells in otherwise healthy adults are heterogeneous and follow distinct virus-specific kinetics. These findings suggests that the distribution of TL and the creation and maintenance of long TL memory T cells could be important for the persistence of long-lived T cell memory.

     

Co-infusion of haplo-identical CD19-chimeric antigen receptor T cells and stem cells achieved full donor engraftment in refractory acute lymphoblastic leukemia

Background

Elderly patients with relapsed and refractory acute lymphoblastic leukemia (ALL) have poor prognosis. Autologous CD19 chimeric antigen receptor-modified T (CAR-T) cells have potentials to cure patients with B cell ALL; however, safety and efficacy of allogeneic CD19 CAR-T cells are still undetermined.

Case presentation

We treated a 71-year-old female with relapsed and refractory ALL who received co-infusion of haplo-identical donor-derived CD19-directed CAR-T cells and mobilized peripheral blood stem cells (PBSC) following induction chemotherapy. Undetectable minimal residual disease by flow cytometry was achieved, and full donor cell engraftment was established. The transient release of cytokines and mild fever were detected. Significantly elevated serum lactate dehydrogenase, alanine transaminase, bilirubin and glutamic-oxalacetic transaminase were observed from days 14 to 18, all of which were reversible after immunosuppressive therapy.

Conclusions

Our preliminary results suggest that co-infusion of haplo-identical donor-derived CAR-T cells and mobilized PBSCs may induce full donor engraftment in relapsed and refractory ALL including elderly patients, but complications related to donor cell infusions should still be cautioned.

Trial registration

Allogeneic CART-19 for Elderly Relapsed/Refractory CD19+ ALL. NCT02799550

     

An unusual case report of indolent T-cell lymphoproliferative disorder with aberrant CD20 expression involving the gastrointestinal tract and bone marrow

Background

Indolent T-cell proliferative disorder of the GIT is a rare and provisional entity in the revised WHO 2016 classification. The patients usually have prolonged survival with persistent disease even without any treatment.

Case presentation

The 46 years old male patient has been followed up for more than 6 years without chemotherapy. Repeated gastrointestinal biopsies showed expansion of the lamina propria extending to the submucosa by small to medium sized lymphocytes with minimal cytologic atypia. The lymphoid cells were positive for CD3, CD43, TIA-1, CD2, CD7 and the B-cell marker CD20; but negative for CD4, CD8, PAX5, CD56, cyclinD1, granzyme (GraB) and Epstein Barr virus-encoded RNA (EBER). Ki-67(MIB1) index was less than 10%. Molecular tests demonstrated a clonal rearrangement for T-cell receptor γ (TCR γ) gene but immunoglobulin chain (IgH, IgK, IgL) gene remained germline. Recognition of possible aberrant CD20 expression in indolent T-cell LPD is important to avoid potential diagnostic pitfall and improper treatment.

Conclusions

We present an unusual case of indolent T-cell lymphoproliferative disorder with aberrant CD20 expression, Recognition of this unusual immunophenotype of indolent T-cell LPD of GI helps to eschew misdiagnosis of B-cell and other high grade lymphomas and inappropriate aggressive treatment.