Increased CTLA‐4+ T cells may contribute to impaired T helper type 1 immune responses in patients with chronic obstructive pulmonary disease

Impaired T helper type 1 (Th1) function is implicated in the susceptibility of patients with chronic obstructive pulmonary disease (COPD) to respiratory infections, which are common causes of acute exacerbations of COPD (AECOPD). To understand the underlying mechanisms, we assessed regulatory T (Treg) cells and the expression of an inhibitory T‐cell receptor, cytotoxic T‐lymphocyte‐associated antigen 4 (CTLA‐4). Cryopreserved peripheral blood mononuclear cells (PBMC) from patients with AECOPD (n = 17), patients with stable COPD (sCOPD; n = 24) and age‐matched healthy non‐smoking controls (n = 26) were cultured for 24 hr with brefeldin‐A or monensin to detect intracellular or surface CTLA‐4 (respectively) by flow cytometry. T cells in PBMC from AECOPD (n = 9), sCOPD (n = 14) and controls (n = 12) were stimulated with anti‐CD3 with and without anti‐CTLA‐4 blocking antibodies and cytokines were quantified by ELISA. Frequencies of circulating T cells expressing intracellular CTLA‐4 were higher in sCOPD (P = 0·01), whereas patients with AECOPD had more T cells expressing surface CTLA‐4 than healthy controls (P = 0·03). Increased frequencies of surface CTLA‐4⁺ CD4⁺ T cells and CTLA‐4⁺ Treg cells paralleled increases in plasma soluble tumour necrosis factor receptor‐1 levels (r = 0·32, P = 0·01 and r = 0·29, P = 0·02, respectively) in all subjects. Interferon‐γ responses to anti‐CD3 stimulation were inversely proportional to frequencies of CD4⁺ T cells expressing intracellular CTLA‐4 (r = −0·43, P = 0·01). Moreover, CTLA‐4 blockade increased the induction of interferon‐γ, tumour necrosis factor‐α and interleukin‐6 in PBMC stimulated with anti‐CD3. Overall, chronic inflammation may expand sub‐populations of T cells expressing CTLA‐4 in COPD patients and therefore impair T‐cell function. CTLA‐4 blockade may restore Th1 function in patients with COPD and so aid the clearance of bacterial pathogens responsible for AECOPD.     

Systemic varicella zoster virus reactive effector memory T‐cells impaired in the elderly and in kidney transplant recipients

Varicella zoster virus (VZV) infections cause varicella and subsequently herpes zoster upon reactivation. Immune‐compromised individuals and the elderly are at high risk of developing herpes zoster due to waning of VZV‐specific T‐cell immunity. In the present study, a novel functional T‐cell assay was developed to test the correlation between age and VZV‐specific T‐cell responses in peripheral blood from healthy individuals. Secondly, VZV‐specific T‐cell responses from renal transplant recipients were compared with healthy individuals. Monocytes were differentiated into mature monocyte‐derived dendritic cells (moDCs) and were infected with VZV. T‐cells were co‐cultured with autologous moDCs infected with VZV and subjected to flowcytometric analysis to identify the phenotype (i.e., naïve [NA: CCR7⁺CD45RO^?], central [CM: CCR7⁺CD45RO⁺] and effector memory [EM: CCR7^?CD45RO⁺] T‐cells) and the frequency of VZV‐reactive T‐cell subsets by intra‐cellular IFN‐γ flowcytometry. In contrast to NA and CM T‐cells, the frequency of VZV‐reactive CD4 and CD8 EM T‐cells was inversely correlated with age (P = 0.0007 and P = 0.01). No difference was found in the percentage of VZV‐reactive CD4 NA, CM and EM T‐cells between transplant recipients and controls. However, the percentage of VZV‐reactive CD8 EM T‐cells was significantly lower in transplant recipients compared to controls (P = 0.02). In conclusion, moDCs infected with VZV are efficient antigen presenting cells applicable to enumerate and characterize the phenotype and differentiation status of the systemic VZV‐specific T‐cell response ex‐vivo. The data suggest that VZV‐reactive EM T‐cells are impaired in the elderly and renal transplant recipients. J. Med. Virol. 84:2018–2025, 2012. © 2012 Wiley Periodicals, Inc.     

Comprehensive T‐cell immunophenotyping and next‐generation sequencing of human papillomavirus (HPV)‐positive and HPV‐negative head and neck squamous cell carcinomas

The success of programmed cell death 1 (PD‐1) inhibition in achieving a clinical response in a subset of head and neck squamous cell carcinoma (HNSCC) patients emphasizes the need to better understand the immunobiology of HNSCC. Immunophenotyping was performed for 30 HCSCC patients [16 human papillomavirus (HPV)‐positive; 14 HPV‐negative] on matched tissue from the primary tumour site, locally metastatic cervical lymph nodes (LNs), uninvolved local cervical LNs, and peripheral blood. CD4⁺ and CD8⁺ T‐cell lymphocytes obtained from tissue were analysed for expression levels of the inhibitory receptors PD‐1, TIM‐3 and CTLA‐4. Next‐generation sequencing of the T‐cell receptor (TCR) β chain was performed on patients (n = 9) to determine receptor repertoire diversity and for clonality analysis. HPV‐negative HNSCC patients, particularly those with stage IV disease, had significantly higher proportions of CD8⁺ T cells expressing CTLA‐4 in tumour tissue (P = 0.0013) and in peripheral blood (P = 0.0344) than HPV‐positive patients, as well as higher expression levels of TIM‐3⁺PD‐1⁺ CD8⁺ T cells (P = 0.0072) than controls. For all patients, PD‐1 expression on CD8⁺ T cells – particularly in HPV‐negative HNSCC cases – strongly correlated (r = 0.63, P = 0.013) with tumour size at the primary site. The top CD8⁺ TCR clones from tumour tissue significantly overlapped with circulating peripheral blood TCR clones (r = 0.946), and HPV‐positive patients had frequently expanded TCR clones that were more hydrophobic – and potentially more immunogenic – than those from HPV‐negative patients. Collectively, our findings demonstrate, for the first time, that high‐stage HPV‐negative HNSCC patients with primary tumours at different sites in the head and neck have elevated peripheral CTLA‐4⁺CD8⁺ T‐cell levels, that tumour‐familiar CD8⁺ T cells are detectable in peripheral blood from HNSCC patients, and that TCRs from HPV‐positive HNSCC patients potentially recognize distinctly immunogenic cognate antigens. However, our findings are preliminary, and need to be further confirmed in a larger patient cohort; also, how these factors affect patient response to immunotherapy needs to be determined. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Abnormal CTLA‐4 function in T cells from patients with systemic lupus erythematosus

CTLA‐4 is a critical gatekeeper of T‐cell activation and immunological tolerance and has been implicated in patients with a variety of autoimmune diseases through genetic association. Since T cells from patients with the autoimmune disease systemic lupus erythematosus (SLE) display a characteristic hyperactive phenotype, we investigated the function of CTLA‐4 in SLE. Our results reveal increased CTLA‐4 expression in FOXP3^? responder T cells from patients with SLE compared with other autoimmune rheumatic diseases and healthy controls. However, CTLA‐4 was unable to regulate T‐cell proliferation, lipid microdomain formation and phosphorylation of TCR‐ζ following CD3/CD28 co‐stimulation, in contrast to healthy T cells. Although lupus T cells responded in vitro to CD3/CD28 co‐stimulation, there was no parallel increase in CTLA‐4 expression, which would normally provide a break on T‐cell proliferation. These defects were associated with exclusion of CTLA‐4 from lipid microdomains providing an anatomical basis for its loss of function. Collectively our data identify CTLA‐4 dysfunction as a potential cause for abnormal T‐cell activation in patients with SLE, which could be targeted for therapy.     

CTLA‐4 (CD152) enhances the Tc17 differentiation program

Although CD8⁺ T cells that produce IL‐17 (Tc17 cells) have been linked to host defense, Tc17 cells show reduced cytotoxic activity, which is the characteristic function of CD8⁺ T cells. Here, we show that CTLA‐4 enhances the frequency of IL‐17 in CD8⁺ T cells, indicating that CTLA‐4 (CD152) specifically promotes Tc17 differentiation. Simultaneous stimulation of CTLA‐4^+/+ and CTLA‐4^?/? T cells in cocultures and agonistic CTLA‐4 stimulation unambiguously revealed a cell‐intrinsic mechanism for IL‐17 control by CTLA‐4. The quality of CTLA‐4‐induced Tc17 cells was tested in vivo, utilizing infection with the facultative intracellular bacterium Listeria monocytogenes (LM). Unlike CTLA‐4^+/+ Tc17 cells, CTLA‐4^?/? were nearly as efficient as Tc1 CTLA‐4^+/+ cells in LM clearance. Additionally, adoptively transferred CTLA‐4^?/? Tc17 cells expressed granzyme B after rechallenge, and produced Tc1 cytokines such as IFN‐γ and TNF‐α, which strongly correlate with bacterial clearance. CTLA‐4^+/+ Tc17 cells demonstrated a high‐quality Tc17 differentiation program ex vivo, which was also evident in isolated IL‐17‐secreting Tc17 cells, with CTLA‐4‐mediated enhanced upregulation of Tc17‐related molecules such as IL‐17A, RORγt, and IRF‐4. Our results show that CTLA‐4 promotes Tc17 differentiation that results in robust Tc17 responses. Its inactivation might therefore represent a central therapeutic target to enhance clearance of infection.     

High‐throughput sequencing of TCR repertoires in multiple sclerosis reveals intrathecal enrichment of EBV‐reactive CD8+ T cells

Epstein‐Barr virus (EBV) has long been suggested as a pathogen in multiple sclerosis (MS). Here, we used high‐throughput sequencing to determine the diversity, compartmentalization, persistence, and EBV‐reactivity of the T‐cell receptor (TCR) repertoires in MS. TCR‐β genes were sequenced in paired samples of cerebrospinal fluid (CSF) and blood from patients with MS and controls with other inflammatory neurological diseases. The TCR repertoires were highly diverse in both compartments and patient groups. Expanded T‐cell clones, represented by TCR‐β sequences >0.1%, were of different identity in CSF and blood of MS patients, and persisted for more than a year. Reference TCR‐β libraries generated from peripheral blood T cells reactive against autologous EBV‐transformed B cells were highly enriched for public EBV‐specific sequences and were used to quantify EBV‐reactive TCR‐β sequences in CSF. TCR‐β sequences of EBV‐reactive CD8⁺ T cells, including several public EBV‐specific sequences, were intrathecally enriched in MS patients only, whereas those of EBV‐reactive CD4⁺ T cells were also enriched in CSF of controls. These data provide evidence for a clonally diverse, yet compartmentalized and persistent, intrathecal T‐cell response in MS. The presented strategy links TCR sequence to intrathecal T‐cell specificity, demonstrating enrichment of EBV‐reactive CD8⁺ T cells in MS.     

Modulation of pulmonary DC function by vaccine‐encoded GM‐CSF enhances protective immunity against Mycobacterium tuberculosis infection

The rational design of new vaccines engineered to target key components of the host immune response is crucial to aid control of important infectious diseases such as tuberculosis. In this report, we determined whether modifying the function of pulmonary APC could improve protection against infection with Mycobacterium tuberculosis. Targeted delivery to the lung of the cytokine GM‐CSF, expressed by the Mycobacterium bovis BCG vaccine strain, increased pulmonary DC numbers and secretion of the immunoregulatory cytokine IL‐12, compared with parental BCG immunization. This impact on APC number by BCG:GM‐CSF resulted in accelerated priming of antigen‐specific CD4⁺ T cells in the mediastinal lymph nodes and increased migration of activated CD4⁺ T cells into the lung. i.n. administration of BCG:GM‐CSF resulted in significantly increased protection against M. tuberculosis infection compared with mice vaccinated with BCG alone. BCG:GM‐CSF exhibited an improved safety profile, as immunodeficient RAG1^?/? mice vaccinated i.n. with BCG:GM‐CSF survived significantly longer than control BCG‐vaccinated mice. These data demonstrate that manipulating immune cells in the lung by BCG‐based delivery of GM‐CSF can assist the development of protective mucosal immunity against pulmonary bacterial infection.     

Local and systemic effects of co‐stimulatory blockade using cytotoxic T lymphocyte antigen‐4‐immunoglobulin in dinitrofluorobenzene‐ and oxazolone‐induced contact hypersensitivity in mice

Cytotoxic T lymphocyte‐associated antigen‐4 (CTLA‐4)‐immunoglobulin (Ig) has immunosuppressive properties both in vivo and in vitro, but much is still unknown about the mechanisms by which CTLA‐4‐Ig exerts its immunosuppressive activities in vivo. The aim of this study was to investigate the effect of CTLA‐4‐Ig in a mouse model of contact hypersensitivity (CHS). The inflammatory response in the presence or absence of CTLA‐4‐Ig was evaluated by measuring the increase in ear thickness in sensitized animals after challenge. We observed a dose‐dependent suppression of the ear swelling in both dinitrofluorobenzene (DNFB)‐ and oxazolone‐induced CHS. The suppressive effect was still present 3 weeks after administration, even in the absence of circulating levels of CTLA‐4‐Ig. It was further shown that CTLA‐4‐Ig inhibits activation of T cells in the draining lymph node after sensitization and affects the maturation level of both dendritic cells and B cells. Furthermore, CTLA‐4‐Ig reduces infiltration of activated CD8⁺ T cells into the inflamed ear tissue and suppresses both local and systemic inflammation, as illustrated by reduced expression of cytokines and chemokines in the inflamed ear and a reduced level of acute‐phase proteins in circulation. Finally, our results suggest that CTLA‐4‐Ig has a mainly immunosuppressive effect during the sensitization phase. We conclude that CTLA‐4‐Ig induces long‐term immunosuppression of both DNFB‐ and oxazolone‐induced inflammation and our data are the first to compare the effect of this compound in both DNFB‐ and oxazolone‐induced CHS and to show that CTLA‐4‐Ig exerts an immunosuppressive effect on both local and systemic inflammatory mediators which is mediated principally during the sensitization phase.     

CTLA‐4 is required by CD4+CD25+ Treg to control CD4+ T‐cell lymphopenia‐induced proliferation

CTLA‐4 is constitutively expressed by CD4⁺CD25⁺Foxp3⁺ Treg but its precise role in Treg function is not clear. Although blockade of CTLA‐4 interferes with Treg function, studies using CTLA‐4‐deficient Treg have failed to reveal an essential requirement for CTLA‐4 in Treg suppression in vivo. Conditional deletion of CTLA‐4 in Foxp3⁺ T cells disrupts immune homeostasis in vivo but the immune processes disrupted by CTLA‐4 deletion have not been determined. We demonstrate that Treg expression of CTLA‐4 is essential for Treg control of lymphopenia‐induced CD4 T‐cell expansion. Despite IL‐10 expression, CTLA‐4‐deficient Treg were unable to control the expansion of CD4⁺ target cells in a lymphopenic environment. Moreover, unlike their WT counterparts, CTLA‐4‐deficient Treg failed to inhibit cytokine production associated with homeostatic expansion and were unable to prevent colitis. Thus, while Treg developing in the absence of CTLA‐4 appear to acquire some compensatory suppressive mechanisms in vitro, we identify a non‐redundant role for CTLA‐4 in Treg function in vivo.     

CD8+ T cells producing IL‐3 and IL‐5 in non‐IgE‐mediated eosinophilic diseases

The cytokines IL‐5, IL‐3, and GM‐CSF are crucial for eosinophil development, survival, and function. To better understand their role in non‐IgE‐mediated eosinophilic diseases, we investigated plasma levels of these cytokines as well as cytokine expression in peripheral blood T cells. While we did not find any evidence for an involvement of T‐cell‐derived GM‐CSF, some of these patients did show an increased proportion of IL‐5‐ or IL‐3‐producing CD4⁺ T cells. However, in a significant proportion of patients, IL‐5‐producing CD8⁺ T cells, so‐called Tc2 cells, which in healthy donors can only be detected at very low levels, were prominent. Furthermore, increased IL‐3 production by CD8⁺ T cells was also observed, strongly supporting the notion that CD8⁺ T cells, not just CD4⁺ T cells, must also be considered as a potential source of the cytokines promoting eosinophilia.     

Pathogenic IL‐23 signaling is required to initiate GM‐CSF‐driven autoimmune myocarditis in mice

Using a mouse model of experimental autoimmune myocarditis (EAM), we showed for the first time that IL‐23 stimulation of CD4⁺ T cells is required only briefly at the initiation of GM‐CFS‐dependent cardiac autoimmunity. IL‐23 signal, acting as a switch, turns on pathogenicity of CD4⁺ T cells, and becomes dispensable once autoreactivity is established. Il23a^?/? mice failed to mount an efficient Th17 response to immunization, and were protected from myocarditis. However, remarkably, transient IL‐23 stimulation ex vivo fully restored pathogenicity in otherwise nonpathogenic CD4⁺ T cells raised from Il23a^?/? donors. Thus, IL‐23 may no longer be necessary to uphold inflammation in established autoimmune diseases. In addition, we demonstrated that IL‐23‐induced GM‐CSF mediates the pathogenicity of CD4⁺ T cells in EAM. The neutralization of GM‐CSF abrogated cardiac inflammation. However, sustained IL‐23 signaling is required to maintain IL‐17A production in CD4⁺ T cells. Despite inducing inflammation in Il23a^?/? recipients comparable to wild‐type (WT), autoreactive CD4⁺ T cells downregulated IL‐17A production without persistent IL‐23 signaling. This divergence on the controls of GM‐CSF‐dependent pathogenicity on one side and IL‐17A production on the other side may contribute to the discrepant efficacies of anti‐IL‐23 therapy in different autoimmune diseases.     

Deletional tolerance prevents AQP4‐directed autoimmunity in mice

Neuromyelitis optica (NMO) is an autoimmune disorder of the central nervous system (CNS) mediated by antibodies to the water channel protein AQP4 expressed in astrocytes. The contribution of AQP4‐specific T cells to the class switch recombination of pathogenic AQP4‐specific antibodies and the inflammation of the blood–brain barrier is incompletely understood, as immunogenic naturally processed T‐cell epitopes of AQP4 are unknown. By immunizing Aqp4 ^?/? mice with full‐length murine AQP4 protein followed by recall with overlapping peptides, we here identify AQP4(201‐220) as the major immunogenic IA^b‐restricted epitope of AQP4. We show that WT mice do not harbor AQP4(201–220)‐specific T‐cell clones in their natural repertoire due to deletional tolerance. However, immunization with AQP4(201–220) of Rag1 ^?/? mice reconstituted with the mature T‐cell repertoire of Aqp4 ^?/? mice elicits an encephalomyelitic syndrome. Similarly to the T‐cell repertoire, the B‐cell repertoire of WT mice is “purged” of AQP4‐specific B cells, and robust serum responses to AQP4 are only mounted in Aqp4 ^?/? mice. While AQP4(201–220)‐specific T cells alone induce encephalomyelitis, NMO‐specific lesional patterns in the CNS and the retina only occur in the additional presence of anti‐AQP4 antibodies. Thus, failure of deletional T‐cell and B‐cell tolerance against AQP4 is a prerequisite for clinically manifest NMO.     

What did we learn from CTLA‐4 insufficiency on the human immune system?

Cytotoxic‐T‐lymphocyte‐antigen‐4 (CTLA‐4) is a negative immune regulator constitutively expressed on regulatory T (Treg) cells and upregulated on activated T cells. CTLA‐4 inhibits T cell activation by various suppressive functions including competition with CD28, regulation of the inhibitory function of Treg cells, such as transendocytosis, and the control of adhesion and motility. Intrinsic CTLA‐4 signaling has been controversially discussed, but so far no distinct signaling pathway has been identified. The CTLA‐4‐mediated Treg suppression plays an important role in the maintenance of peripheral tolerance and the prevention of autoimmune diseases. Human CTLA‐4 insufficiency is caused by heterozygous germline mutations in CTLA4 and characterized by a complex immune dysregulation syndrome. Clinical studies on CTLA4 mutation carriers showed a reduced penetrance and variable expressivity, suggesting modifying factor(s). One hundred and forty‐eight CTLA4 mutation carriers have been reported; patients showed hypogammaglobulinemia, recurrent infectious diseases, various autoimmune diseases, and lymphocytic infiltration into multiple organs. The CTLA‐4 expression level in Treg cells was reduced, while the frequency of Treg cells was increased in CTLA‐4‐insufficient patients. The transendocytosis assay is a specific functional test for the assessment of newly identified CTLA4 gene variants. Immunoglobulin substitution, corticosteroids, immunosuppressive therapy, and targeted therapy such as with CTLA‐4 fusion proteins and mechanistic target of rapamycin (mTOR) inhibitors were applied; patients with life‐threatening, treatment‐resistant symptoms underwent hematopoietic stem cell transplantation. The fact that in humans CTLA‐4 insufficiency causes severe disease taught us that the amount of CTLA‐4 molecules present in/on T cells matters for immune homeostasis. However, whether the pathology‐causing activated T lymphocytes in CTLA‐4‐insufficient patients are antigen‐specific is an unsolved question. CTLA‐4, in addition, has a role in autoimmune diseases and cancer. Anti‐CTLA‐4 drugs are employed as checkpoint inhibitors to target various forms of cancer. Thus, clinical research on human CTLA‐4 insufficiency might provide us a deeper understanding of the mechanism(s) of the CTLA‐4 molecule and immune dysregulation disorders.     

Intrathecal CD4+ and CD8+ T‐cell responses to endogenously synthesized candidate disease‐associated human autoantigens in multiple sclerosis patients

MS pathology is potentially orchestrated by autoreactive T cells, but the antigens recognized remain unknown. A novel APC/T‐cell platform was developed to determine intrathecal CD4⁺ and CD8⁺ T‐cell responses to candidate MS‐associated autoantigens (cMSAg) in clinically isolated syndrome (CIS, n = 7) and MS (n = 6) patients. Human cMSAg encoding open reading frames (n = 8) were cloned into an Epstein–Barr virus (EBV)‐based vector to express cMSAg at high levels in EBV‐transformed B‐cells (BLCLs). Human cMSAg cloned were myelin‐associated and ‐oligodendrocyte glycoprotein, myelin basic protein, proteolipid protein, ATP‐dependent potassium channel ATP‐dependent inwards rectifying potassium channel 4.1, S100 calcium‐binding protein B, contactin‐2, and neurofascin. Transduced BLCLs were used as autologous APC in functional T‐cell assays to determine cMSAg‐specific T‐cell frequencies in cerebrospinal fluid derived T‐cell lines (CSF‐TCLs) by intracellular IFN‐γ flow cytometry. Whereas all CSF‐TCL responded strongly to mitogenic stimulation, no substantial T‐cell reactivity to cMSAg was observed. Contrastingly, measles virus fusion protein‐specific CD4⁺ and CD8⁺ T‐cell clones, used as control of the APC/T‐cell platform, efficiently recognized transduced BLCL expressing their cognate antigen. The inability to detect substantial T‐cell reactivity to eight human endogenously synthesized cMSAg in autologous APC do not support their role as prominent intrathecal T‐cell target antigens in CIS and MS patients early after onset of disease.     

Fasciola hepatica tegumental antigens induce anergic‐like T cells via dendritic cells in a mannose receptor‐dependent manner

FoxP3⁺ Treg cells and anergic T cells are the two regulatory phenotypes of T‐cell responses associated with helminth infection. Here, we examine the T‐cell responses in mice during Fasciola hepatica infection, and to its tegumental coat antigens (FhTeg) that are shed from the fluke every 2–3 h. FhTeg comprises a rich source of glycoproteins, mainly oligomannose N‐glycans that bind to mannose receptor. This study demonstrated a novel mechanism for the T‐cell unresponsiveness observed during F. hepatica infection and after injection with FhTeg. Markers of T‐cell anergy, such as GRAIL, EGR2, ICOS, and ITCH, are enhanced amongst CD4⁺ T‐cell populations during infection and following FhTeg injection. This is characterized by a lack of cytokine responses and reduced proliferative activity, which can be reversed with the addition of IL‐2. FhTeg‐activated dendritic cells (DCs) suppress T cells in vitro as measured by enhanced GRAIL and CTLA4 by RNA and suppressed cytokine expression in anti‐CD3 stimulated CD4⁺ T cells. FhTeg‐treated DCs have enhanced MR expression, which is critical for DC‐CD4⁺ T‐cell communication. Taken together, this study presents markers of anergy in a mouse model of F. hepatica infection, and improves our understanding of host–pathogen interactions and how helminths modulate host immunity.     

Different interleukin‐17‐secreting Toll‐like receptor+ T‐cell subsets are associated with disease activity in multiple sclerosis

Signalling through Toll‐like receptors (TLRs) may play a role in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS). In the present study, the expression of TLR‐2, ‐4 and ‐9 was significantly higher on CD4⁺ and CD8⁺ T‐cells from MS patients compared to healthy individuals. Following in‐vitro activation, the proportion of interleukin (IL)‐17⁺ and IL‐6⁺ CD4⁺ and CD8⁺ T‐cells was higher in the patients. In addition, the proportion of IFN‐γ‐secreting TLR⁺ CD8⁺ T‐cells was increased in MS patients. Among different IL‐17⁺ T‐cell phenotypes, the proportion of IL‐17⁺ TLR⁺ CD4⁺ and CD8⁺ T‐cells producing IFN‐γ or IL‐6 were positively associated with the number of active brain lesions and neurological disabilities. Interestingly, activation of purified CD4⁺ and CD8⁺ T‐cells with ligands for TLR‐2 (Pam3Csk4), TLR‐4 [lipopolysaccharide (LPS)] and TLR‐9 [oligodeoxynucleotide (ODN)] directly induced cytokine production in MS patients. Among the pathogen‐associated molecular patterns (PAMPs), Pam3Csk4 was more potent than other TLR ligands in inducing the production of all proinflammatory cytokines. Furthermore, IL‐6, IFN‐γ, IL‐17 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) levels produced by Pam3Csk4‐activated CD4⁺ cells were directly associated with disease activity. A similar correlation was observed with regard to IL‐17 levels released by Pam3Csk4‐stimulated CD8⁺ T‐cells and clinical parameters. In conclusion, our data suggest that the expansion of different T helper type 17 (Th17) phenotypes expressing TLR‐2, ‐4 and ‐9 is associated with MS disease activity, and reveals a preferential ability of TLR‐2 ligand in directly inducing the production of cytokines related to brains lesions and neurological disabilities.     

Tumor‐specific CD4+ T cells maintain effector and memory tumor‐specific CD8+ T cells

Immunotherapies that augment antitumor T cells have had recent success for treating patients with cancer. Here we examined whether tumor‐specific CD4⁺ T cells enhance CD8⁺ T‐cell adoptive immunotherapy in a lymphopenic environment. Our model employed physiological doses of tyrosinase‐related protein 1‐specific CD4⁺ transgenic T cells‐CD4⁺ T cells and pmel‐CD8⁺ T cells that when transferred individually were subtherapeutic; however, when transferred together provided significant (p ≤ 0.001) therapeutic efficacy. Therapeutic efficacy correlated with increased numbers of effector and memory CD8⁺ T cells with tumor‐specific cytokine expression. When combined with CD4⁺ T cells, transfer of total (naïve and effector) or effector CD8⁺ T cells were highly effective, suggesting CD4⁺ T cells can help mediate therapeutic effects by maintaining function of activated CD8⁺ T cells. In addition, CD4⁺ T cells had a pronounced effect in the early posttransfer period, as their elimination within the first 3 days significantly (p < 0.001) reduced therapeutic efficacy. The CD8⁺ T cells recovered from mice treated with both CD8⁺ and CD4⁺ T cells had decreased expression of PD‐1 and PD‐1‐blockade enhanced the therapeutic efficacy of pmel‐CD8 alone, suggesting that CD4⁺ T cells help reduce CD8⁺ T‐cell exhaustion. These data support combining immunotherapies that elicit both tumor‐specific CD4⁺ and CD8⁺ T cells for treatment of patients with cancer.     

Programmed death (PD)‐1 molecule and its ligand PD‐L1 distribution among memory CD4 and CD8 T cell subsets in human immunodeficiency virus‐1‐infected individuals

Human immunodeficiency virus (HIV)‐1 causes T cell anergy and affects T cell maturation. Various mechanisms are responsible for impaired anti‐HIV‐1‐specific responses: programmed death (PD)‐1 molecule and its ligand PD‐L1 are negative regulators of T cell activity and their expression is increased during HIV‐1 infection. This study examines correlations between T cell maturation, expression of PD‐1 and PD‐L1, and the effects of their blockade. Peripheral blood mononuclear cells (PBMC) from 24 HIV‐1⁺ and 17 uninfected individuals were phenotyped for PD‐1 and PD‐L1 expression on CD4⁺ and CD8⁺ T cell subsets. The effect of PD‐1 and PD‐L1 blockade on proliferation and interferon (IFN)‐γ production was tested on eight HIV‐1⁺ patients. Naive (CCR7⁺CD45RA⁺) CD8⁺ T cells were reduced in HIV‐1 aviraemic (P = 0·0065) and viraemic patients (P = 0·0130); CD8 T effector memory subsets [CCR7^‐CD45RA^–(T_EM)] were increased in HIV‐1⁺ aviraemic (P = 0·0122) and viraemic (P = 0·0023) individuals versus controls. PD‐1 expression was increased in CD4 naive (P = 0·0496), central memory [CCR7⁺CD45RA^– (T_CM); P = 0·0116], T_EM (P = 0·0037) and CD8 naive T cells (P = 0·0133) of aviraemic HIV‐1⁺versus controls. PD‐L1 was increased in CD4 T_EMRA (CCR7^‐CD45RA⁺, P = 0·0119), CD8 T_EM (P = 0·0494) and CD8 T_EMRA (P = 0·0282) of aviraemic HIV‐1⁺versus controls. PD‐1 blockade increased HIV‐1‐specific proliferative responses in one of eight patients, whereas PD‐L1 blockade restored responses in four of eight patients, but did not increase IFN‐γ‐production. Alteration of T cell subsets, accompanied by increased PD‐1 and PD‐L1 expression in HIV‐1 infection contributes to anergy and impaired anti‐HIV‐1‐specific responses which are not rescued when PD‐1 is blocked, in contrast to when PD‐L1 is blocked, due possibly to an ability to bind to receptors other than PD‐1.     

Lack of Mycobacterium tuberculosis–specific interleukin‐17A–producing CD4+ T cells in active disease

Protective immunity to Mycobacterium tuberculosis (Mtb) is commonly ascribed to a Th1 profile; however, the involvement of Th17 cells remains to be clarified. Here, we characterized Mtb‐specific CD4⁺ T cells in blood and bronchoalveolar lavages (BALs) from untreated subjects with either active tuberculosis disease (TB) or latent Mtb infection (LTBI), considered as prototypic models of uncontrolled or controlled infection, respectively. The production of IL‐17A, IFN‐γ, TNF‐α, and IL‐2 by Mtb‐specific CD4⁺ T cells was assessed both directly ex vivo and following in vitro antigen‐specific T‐cell expansion. Unlike for extracellular bacteria, Mtb‐specific CD4⁺ T‐cell responses lacked immediate ex vivo IL‐17A effector function in both LTBI and TB individuals. Furthermore, Mtb‐specific Th17 cells were absent in BALs, while extracellular bacteria‐specific Th17 cells were identified in gut biopsies of healthy individuals. Interestingly, only Mtb‐specific CD4⁺ T cells from 50% of LTBI but not from TB subjects acquired the ability to produce IL‐17A following Mtb‐specific T‐cell expansion. Finally, IL‐17A acquisition by Mtb‐specific CD4⁺ T cells correlated with the coexpression of CXCR3 and CCR6, currently associated to Th1 or Th17 profiles, respectively. Our data demonstrate that Mtb‐specific Th17 cells are selectively undetectable in peripheral blood and BALs from TB patients.