Critical role of spatial interaction between CD8+ and Foxp3+ cells in human gastric cancer: the distance matters

Purpose

In various cancer types, an abundance of FoxP3⁺ regulatory T cells (Treg) has been associated with an unfavorable outcome. Yet, the role of Treg on cancer immunity has been shown to be complex. In single cell marker technique, other tumor-infiltrating lymphocytes (TILs) such as cytotoxic CD8⁺ T cells (CTL) also influenced prognosis. This study for the first time investigates the concurrent spatial distribution pattern of CD8⁺ and FoxP3⁺ TILs and their prognostic impact in human gastric cancer.

Materials and methods

Tumor tissue microarrays of 50 patients with surgically treated adenocarcinoma of the cardia were studied. An immunohistochemical double staining of CD8⁺ and FoxP3⁺ TILs was performed. Cell counts and cell-to-cell distances in tumor epithelium and stroma were evaluated with image-processing software. Metastasis-free survival, no-evidence-of-disease survival, and overall survival were investigated (mean follow-up time 6.9 years).

Results

High intraepithelial infiltration of CD8⁺ and FoxP3⁺ TIL was associated with the improved 10-year metastasis-free survival (83 vs. 54 %, p = 0.04 and 85 vs. 59 %, p = 0.09, respectively). Considering cell-to-cell distance and comparing patients with functional (30–110 μm) versus nonfunctional distances of CD8⁺ and FoxP3⁺ TILs, 10-year survival rates differed between 89 and 55 % (p = 0.009), respectively.

Conclusion

Prognostic influence of tumor-infiltrating immune cells in gastric cancer critically depends on their cell-to-cell distance. FoxP3⁺ TILs must be located within a distance between 30 and 110 μm of CD8⁺ T cells to positively impact on prognosis.     

CD8 T cells expressing NK associated receptors are increased in melanoma patients and display an effector phenotype

CD8⁺ T cells can express NK-associated receptors (NKRs) that may regulate their cytolytic function. We have characterized the expression of several NKRs on peripheral blood CD8⁺ T cells from melanoma patients and compared them to age-matched healthy donors. The analysis performed includes HLA class I specific receptors (KIRs, LILRB1 and CD94/NKG2) and other NK receptors like CD57, CD56 and CD16. Melanoma patients showed a higher variability in the expression of NKRs on circulating CD8⁺ T cells than age-matched healthy donors. NKR expression on CD8⁺ T cells from melanoma patients showed a significant increase of KIR2DL2/L3/S2 (mAb gl183), CD244, CD57, CD56 and CD16. We have also found an increase of CD8⁺ CD28^– CD27^– T cells in melanoma patients. This subset represents terminally differentiated effector cells expressing CD244 and high levels of perforin. The expression of NKRs was also mainly restricted to this T cell subset. Altogether, circulating CD8⁺ T cells from melanoma patients display a distinct phenotype characterized by downregulation of costimulatory molecules and higher expression of NKRs. We suggest that the increased expression of NKRs on T cells may contribute to the final outcome of the immune response against melanoma both stimulating or inhibiting activation and differentiation to effector cells. Blocking inhibitory receptor function and enhancing activating receptors may represent new strategies with therapeutic potential against melanoma.     

T cell profiling reveals high CD4+CTLA-4+ T cell frequency as dominant predictor for survival after Prostate GVAX/ipilimumab treatment

Immune checkpoint blockade enhances antitumor responses, but can also lead to severe immune-related adverse events (IRAE). To avoid unnecessary exposure to these potentially hazardous agents, it is important to identify biomarkers that correlate with clinical activity and can be used to select patients that will benefit from immune checkpoint blockade. To understand the consequences of CTLA-4 blockade and identify biomarkers for clinical efficacy and/or survival, an exploratory T cell monitoring study was performed in a phase I/II dose escalation/expansion trial (n = 28) of combined Prostate GVAX/ipilimumab immunotherapy. Phenotypic T cell monitoring in peripheral blood before and after Prostate GVAX/ipilimumab treatment revealed striking differences between patients who benefited from therapy and patients that did not. Treatment-induced rises in absolute lymphocyte counts, CD4⁺ T cell differentiation, and CD4⁺ and CD8⁺ T cell activation were all associated with clinical benefit. Moreover, significantly prolonged overall survival (OS) was observed for patients with high pre-treatment frequencies of CD4⁺CTLA-4⁺, CD4⁺PD-1⁺, or differentiated (i.e., non-naive) CD8⁺ T cells or low pre-treatment frequencies of differentiated CD4⁺ or regulatory T cells. Unsupervised clustering of these immune biomarkers revealed cancer-related expression of CTLA-4⁺ in CD4⁺ T cells to be a dominant predictor for survival after Prostate GVAX/ipilimumab therapy and to thus provide a putative and much-needed biomarker for patient selection prior to therapeutic CTLA4 blockade.     

Decreased CD8<Superscript>+</Superscript>T cell response to Epstein-Barr virus infected B cells in multiple sclerosis is not due to decreased HLA class I expression on B cells or monocytes

Background  

Patients with multiple sclerosis (MS) have a decreased frequency of CD8⁺ T cells reactive to their own Epstein-Barr virus (EBV) infected B cells. We have proposed that this might predispose to the development of MS by allowing EBV-infected autoreactive B cells to accumulate in the central nervous system. The decreased CD8⁺ T cell response to EBV results from a general CD8⁺ T cell deficiency and also a decreased proportion of EBV-specific T cells within the total CD8⁺ T cell population. Because decreased HLA class I expression on monocytes and B cells has been reported in MS and could influence the generation and effector function of EBV-specific CD8⁺ T cells, the present study was undertaken to measure the expression of HLA molecules on B cells and monocytes in patients with MS.     

Intratumoral CD8+ T/FOXP3+ cell ratio is a predictive marker for survival in patients with colorectal cancer

The human immune system consists of a balance between immune surveillance against non-self antigens and tolerance of self-antigens. CD8⁺ T cells and CD4⁺ regulatory T cells (Tregs) are the main players for immune surveillance and tolerance, respectively. We examined immunohistochemically the immunological balance at the tumor site using 94 surgically resected colorectal cancer tissues. Forkhead box P3 (FOXP3)⁺ cells were considered to be Tregs in the present study. The number of intratumoral FOXP3⁺ cells (itFOXP3⁺ cells) was positively correlated with lymph node metastases (P = 0.030). itCD8⁺ T/itFOXP3⁺ cell ratio negatively correlated with pathological stages (P = 0.048). Next, relationship between the number of itCD8⁺ T cells or itFOXP3⁺ cells and survival prognosis in 94 patients who underwent a curative resection was analyzed. Only itCD8⁺ T/itFOXP3⁺ cell ratio positively correlated with disease-free survival (0.023) and overall survival (P = 0.010). Multivariate analysis indicated that itCD8⁺ T/itFOXP3⁺ cell ratio is an independent prognostic factor (P = 0.035) of overall survival. The number of itFOXP3⁺ cells positively correlated with transforming growth factor-beta TGF-β production at the tumor site (P = 0.020). In conclusion, itCD8⁺ T/itFOXP3⁺ cell ratio is a predictive marker for both disease-free survival time and overall survival time in patients with colorectal cancer. Importantly, itCD8⁺ T/itFOXP3⁺ cell ratio may be an independent prognostic factor. And, tumor-producing TGF-β may contribute to the increased number of itFOXP3⁺ cells.     

TGF-β modulates the functionality of tumor-infiltrating CD8<Superscript>+</Superscript> T cells through effects on TCR signaling and Spred1 expression

This study demonstrates that CD8⁺ T cells in the tumor microenvironment display reduced functionality and hyporesponsiveness. TGF-β contributed markedly to the tumor-infiltrating CD8⁺ T cells’ (TILs) reduced functionality, which could be reversed using a small molecule TGF-β inhibitor. Upon T-cell receptor (TCR) activation, the activation of ITK and ERK kinases were reduced in CD8⁺ TILs, as compared to splenic CD8⁺ T cells: TGF-β inhibitor could reverse this phenomenon. This study demonstrates for the first time the association of the Spred-1 gene, an inhibitor of the Ras/MAPK pathway, with CD8⁺ TILs and TGF-β activity. Spred-1 was upregulated in CD8⁺ TILs and TGF-β enhanced the expression of Spred-1 in effector/memory CD8⁺ T cells and not in rested/memory CD8⁺ T cells. Based on these findings, this study supports the hypothesis that TGF-β mediates an inhibitory mechanism on CD8⁺ TILs involving TCR-signaling blockade and the upregulation of Spred-1, thus implicating Spred-1 as a potential new target for future anti-tumor immune studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. M. G. di Bari and M. E. C. Lutsiak contributed equally to this work.     

Expression of Treg Subsets on Intestinal T Cell Immunity and Endotoxin Translocation in Porcine Sepsis After Severe Burns

In the present study, the changes of the regulatory T cells (Treg) expression, endotoxin translocation, and the relationships in intestinal lymph nodes were investigated in porcine sepsis induced by severe burns. Flow cytometry, western blot, and Tachypleus amebocyte lysate were applied to study after the burn injury model was built. We found that the upregulated Treg expression was negatively related to the CD3⁺CD4⁺/CD3⁺CD8⁺ ratio (r = ?0.832, P < 0.05) after burn injury-induced sepsis. While Treg expression and portal venous plasma endotoxin translocation levels were positively correlated (r = 0.876, P < 0.05) when compared with the control group. Moreover, we detected a transforming of T cell subsets from T helper 1 cells to T helper 2 cells. Therefore, intestinal Treg cells expression exerts immunosuppressive effects on other intestinal T lymphocytes and was closely related to endotoxin translocation in porcine sepsis after severe burns injuries. Above all, the intestinal Treg cells may play an important role in the intestinal immune barrier system after severe burns injuries.     

Peripheral CD8+ T cell proliferation is prognostic for patients with advanced thoracic malignancies

There is a complex interplay between the immune system and a developing tumor that is manifest in the way that the balance of T cell subsets in the local tumor environment reflects clinical outcome. Tumor infiltration by CD8⁺ T cells and regulatory T cells (Treg) is associated with improved and reduced survival, respectively, in many cancer types. However, little is known of the prognostic value of immunological parameters measured in peripheral blood. In this study, peripheral CD8⁺ T cells and Treg from 43 patients with malignant mesothelioma or advanced non-small-cell lung cancer scheduled to commence palliative chemotherapy were assessed by flow cytometry and evaluated for association with patient survival. Patients had a higher proportion of peripheral Treg, proliferating CD8⁺ T cells and CD8⁺ T cells with an activated effector phenotype compared with age-matched healthy controls. Higher proportions of Treg and proliferating CD8⁺ T cells were both associated with poor survival in univariate analyses (hazard ratio [HR] 3.81, 95 % CI 1.69–8.57; p < 0.01 and HR 2.86, 95 % CI 1.26–6.50; p < 0.05, respectively). CD8⁺ T cell proliferation was independently predictive of reduced survival in multivariate analysis (HR 2.58, 95 % CI 1.01–6.61; p < 0.05). These findings suggest that peripheral CD8⁺ T cell proliferation can be a useful prognostic marker in patients with thoracic malignancies planned for palliative chemotherapy.     

Identification and HLA-Tetramer-Validation of Human CD4+ and CD8+ T Cell Responses against HCMV Proteins IE1 and IE2

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4⁺ and CD8⁺ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8⁺ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4⁺ and CD8⁺ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4⁺ and CD8⁺ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4⁺ and 44 CD8⁺ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy.     

Higher numbers of T-bet+ intratumoral lymphoid cells correlate with better survival in gastric cancer

In the present study, we studied the expression of T-bet, a key marker for type 1 immune responses, within the tumor microenvironment of gastric cancer, and analyzed its association with clinicopathological parameters. One hundred and fifty-two archival paraffin-embedded gastric tumor tissues were collected, and the expression of T-bet in these cancer tissue specimens was examined by immunohistochemistry. T-bet⁺ tumor-infiltrating lymphocytes (TILs) in some gastric cancer tissues were further characterized by flow cytometric analysis. The density of T-bet⁺ TILs in gastric cancer tissues in relation to patient’s clinicopathological parameters and postoperative prognosis has been analyzed. Herein, we have found significant increases in T-bet⁺ lymphocytes in tumor tissues as compared with normal stomach tissues, gastritis tissues or gastric polyp specimens. T-bet⁺ cells mainly consisted of CD4⁺, CD8⁺ and CD56⁺ TILs. In addition, lower numbers of T-bet⁺ TILs were associated with poor clinicopathological parameters such as invasion to muscular layer, larger tumor size and advanced cancer stages. Moreover, patients with higher numbers of T-bet⁺ TILs have longer disease-free survival and overall survival. Thus, our study supports the idea that tumor growth elicits spontaneous type 1 cellular immune responses and tumor progression is associated with suppression of antitumor immunity. T-bet expression within tumor can serve as a prognostic indicator for gastric cancer and a potential biomarker for immunotherapy.     

Elevated level of peripheral CD8+CD28− T lymphocytes are an independent predictor of progression-free survival in patients with metastatic breast cancer during the course of chemotherapy

Purpose

Suppression of cellular immunity resulting from tumorigenesis and/or therapy might promote cancer cells’ growth, progression and invasion. Here, we explored whether T lymphocyte subtypes from peripheral blood of metastatic breast cancer (MBC) female patients could be used as alternative surrogate markers for cancer progress. Additionally, plasma levels of interleukin (IL)-2, IL-4, IL-6, IL-10, IFN-γ, and transforming growth factor-β1 were quantitated from MBC and healthy volunteers.

Experimental design

This study included 89 female MBC patients during the post-salvage chemotherapy follow-up and 50 age- and sex-matched healthy volunteers as control. The percentages of T lymphocyte subpopulations from peripheral blood and plasma levels of cytokines were measured.

Results

Both CD8⁺CD28^? and CD4⁺CD25⁺ were elevated in MBC patients compared to the control cohort (P < 0.05). In contrast, CD3⁺ and CD8⁺CD28⁺cells were significantly lower in MBC patients (P < 0.0001, P = 0.045, respectively). MBC patients had elevated levels of immunosuppressive cytokines IL-6 and IL-10. Patients with elevated CD8⁺CD28^? and CD4⁺CD25⁺ cells showed increased levels of IL-6, and only patients with elevated CD8⁺CD28^? had decreased interferon-γ. Univariate analysis indicated increased CD3⁺CD4⁺ or CD8⁺CD28⁺correlated with prolonged progression-free survival (PFS), while elevated CD8⁺CD28^?associated with shorten PFS. The percent of CD8⁺CD28^? T lymphocytes is an independent predictor for PFS through multivariate analysis.

Conclusions

This study suggests that progressive elevated levels of CD8⁺CD28^? suppressor T lymphocytes represent a novel independent predictor of PFS during post-chemotherapy follow-up.     

Decreased expression of interleukin-36α correlates with poor prognosis in hepatocellular carcinoma

Interleukin-36α (IL-36α) has been found to have a prominent role in the pathogenesis of inflammatory disorders; however, little is known about the role of IL-36α in cancer. In this study, we investigated the expression, prognostic value, and the underlying antitumor mechanism of IL-36α in hepatocellular carcinoma (HCC). From immunohistochemistry analysis, IL-36α expression was lower in poorly differentiated HCC cells. In clinicopathological analysis, low IL-36α expression significantly correlated with tumor size, histological differentiation, tumor stage, and vascular invasion, and low intratumoral IL-36α expression had significantly worse overall survival rates and shorter disease-free survival rates. Moreover, intratumoral IL-36α expression was an independent risk factor for overall survival. Consecutive sections were used to detect CD3⁺, CD8⁺, and CD4⁺ tumor-infiltrating lymphocytes (TILs), and we found that high-IL-36α-expressing tumor tissues exhibited a significantly higher proportion of intratumoral CD3⁺ and CD8⁺ TILs, but not CD4⁺ TILs. Our in vitro model confirmed that supernatant from IL-36α-overexpressing human HCC cells had an increased capacity to recruit CD3⁺ and CD8⁺ T cells. Consistently, mouse HCC cells engineered to overexpress IL-36α demonstrated markedly delayed growth in vivo, as well as higher levels of intratumoral CD3⁺ and CD8⁺ TILs, compared with control mice. In vitro chemotaxis analysis also showed that mouse HCC cells overexpressing IL-36α could recruit more number of CD3⁺ and CD8⁺ T cells. These results show that IL-36α expression may play a pivotal role in determining the prognosis of patients with HCC, which we attribute to the activation of adaptive T cell immunity, especially CD8⁺ T cell immune response.     

HCV monoinfection and HIV/HCV coinfection enhance T-cell immune senescence in injecting drug users early during infection

Background

Injecting drug users (IDU) are at premature risk of developing multimorbidity and mortality from causes commonly observed in the elderly. Ageing of the immune system (immune-senescence) can lead to premature morbidity and mortality and can be accelerated by chronic viral infections. Here we investigated the impact of HCV monoinfection and HIV/HCV coinfection on immune parameters in (ex-) IDU. We analyzed telomere length and expression of activation, differentiation and exhaustion markers on T cells at baseline (t?=?1) and at follow-up (t?=?2) (median interval 16.9 years) in IDU who were: HCV mono-infected (n?=?21); HIV/HCV coinfected (n?=?23) or multiple exposed but uninfected (MEU) (n?=?8).

Results

The median time interval between t?=?1 and t?=?2 was 16.9 years. Telomere length within CD4⁺ and CD8⁺ T cells decreased significantly over time in all IDU groups (p?≤?0.012). CD4⁺ T-cell telomere length in HCV mono-infected IDU was significantly reduced compared to healthy donors at t?=?1 (p?<?0.008). HIV/HCV coinfected IDU had reduced CD4⁺ and CD8⁺ T-cell telomere lengths (p?≤?0.002) to healthy donors i at t?=?1. This was related to persistent levels of immune activation but not due to increased differentiation of T cells over time. Telomere length decrease was observed within all T-cell subsets, but mainly found in immature T cells (CD27⁺CD57⁺) (p?≤?0.015).

Conclusions

HCV mono-infection and HIV/HCV coinfection enhance T-cell immune-senescence. Our data suggest that this occurred early during infection, which warrants early treatment for both HCV and HIV to reduce immune senescence in later life.

     

Increased prevalence of tumour infiltrating immune cells in oropharyngeal tumours in comparison to other subsites: relationship to peripheral immunity

Background

The nature of the tumour microenvironment immune response in head and neck cancer patients has an important role in tumour development and metastasis, but it is unknown if this differs between cancer subsites or whether it is related to the peripheral immune response.

Methods

Immune cells (CD4, CD8, Foxp3) in head and neck squamous cell carcinoma tissue (HNSCC; n = 66), detected by immunohistochemistry, have been correlated with tumour subsite and immune cells in the peripheral circulation (CD4⁺CD25^HighFoxp3⁺ Treg and CD4⁺ T cells), identified using flow cytometry.

Results

Oropharyngeal tumours had a greater number of infiltrating immune cells in both tumour and stroma compared with other subsites, but no difference was observed in the circulating levels. Immune cells in the stroma were positively related to those in the tumour with consistently higher levels in stroma. A strong relationship was found between the number of CD4⁺ and Foxp3⁺ cells but not between the number of CD8⁺ and Foxp3⁺ cells in the tumour. The number of Foxp3⁺ cells within the tumour was positively correlated with the percentage of circulating CD4⁺CD25^High cells positive for Foxp3. Late stage laryngeal tumours showed a higher number of Foxp3⁺ lymphocytes compared with early stage malignancies, and oropharyngeal tumours had more CD4⁺ cells in node negative tumours compared with node positive ones.

Conclusion

The level of immune cell infiltration in head and neck squamous cell carcinoma appears to be subsite dependent residing primarily in the stroma and is likely to be dependent on the peripheral immune response.     

TLR8 stimulation enhances cetuximab-mediated natural killer cell lysis of head and neck cancer cells and dendritic cell cross-priming of EGFR-specific CD8+ T cells

Background

Cetuximab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody that prolongs survival in the treatment for head and neck cancer (HNC), but only in 10–20 % of patients. An immunological mechanism of action such as natural killer (NK) cell–mediated antibody-dependent cellular cytotoxicity (ADCC) has been suggested. We investigated the effects of activating toll-like receptor (TLR)-8 to enhance activity of cetuximab-stimulated, FcγR-bearing cells.

Objective

To determine the capability of TLR8-stimulation to enhance the activation and function of NK cells and dendritic cells (DC) in the presence of cetuximab-coated HNC cells.

Methods

Peripheral blood mononuclear cells (PBMC), NK, DC, and CD8⁺ T cells were isolated and analyzed using ⁵¹Cr release ADCC, flow cytometry analysis, cytokine ELISA, and EGFR_853-861 tetramer staining.

Results

TLR8 stimulation of unfractionated PBMC led to enhanced cetuximab-mediated ADCC in healthy donors (p < 0.01) and HNC patients (p < 0.001), which was dependent on NK cells. Secretion of Th1 cytokines TNFα (p < 0.0001), IFNγ (p < 0.0001), and IL-12p40 (p < 0.005) was increased. TLR8 stimulation of PBMC augmented cetuximab-enhanced NK cell degranulation (p < 0.001). TLR8-stimulated NK cells enhanced DC maturation markers CD80, CD83, and CD86 in co-culture with cetuximab-treated HNC cells. TLR8 stimulation of NK-DC co-cultures significantly increased DC priming of EGFR-specific CD8⁺ T cells in the presence of cetuximab.

Discussion

VTX-2337 and cetuximab combination therapy can activate innate and adaptive anti-cancer immune responses. Further investigation in human trials will be important for determining the clinical benefit of this combination and for determining biomarkers of response.     

Tumour infiltrating lymphocytes correlate with improved survival in patients with oesophageal adenocarcinoma

Background

Oesophageal adenocarcinoma (OAC) is increasingly common in the west, and survival remains poor at 10–15 % at 5 years. Immune responses are increasingly implicated as a determining factor of tumour progression. The ability of lymphocytes to recognise tumour antigens provides a mechanism for a host immune attack against cancer providing a potential treatment strategy.

Materials and Methods

Tumour infiltrating lymphocytes (TILs: CD3+, CD4+, CD8+ and FOXp3+) were assessed by immunohistochemistry using tissue microarrays in a contemporary and homogeneous cohort of OAC patients (n = 128) undergoing curative treatment.

Results

Multivariate analysis identified three independent prognostic factors for improved cancer-specific survival (CSS): increased CD8+ TILs (p = 0.003), completeness of resection (p < 0.0001) and lower pathological N stage (p < 0.0001). Independent prognostic factors for favourable disease-free survival included surgery-only treatment (p = 0.015), completeness of resection (p = 0.001), increased CD8+ TILs (p < 0.0001) and reduced pathological N stage (p < 0.0001). Higher levels of TILs in the pathological specimen were associated with significant pathological response to neoadjuvant chemotherapy (NAC). On multivariate analysis increased levels of CD4+ (p = 0.017) and CD8+ TILs (p = 0.005) were associated with significant local tumour regression and lymph node downstaging, respectively.

Discussion

Our results establish an association of TILs and survival in OAC, as seen in other solid tumours, and identify particular TIL subsets that are present at higher levels in patients who responded to NAC compared to non-responders. These findings highlight potential therapeutic strategies in EAC based on utilising the host immunological response and highlight the immune responses biomarker potential.

     

Tuberculosis patients display a high proportion of CD8+ T cells with a high cytotoxic potential

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) and remains a major cause of morbidity and mortality worldwide. In the host's immune response system, T cells play a critical role in mediating protection against Mtb infection, but the role of CD8⁺ T cells is still controversial. We evaluated the phenotypical characterization and cytotoxic ability of CD8⁺ T cells by flow cytometry‐based assay. Cytokine levels in serum were measured by multiplex cytokine assay. Our data show that cells from TB patients have an increased percentage of peripheral blood CD8⁺αβ⁺ T (p = 0.02) and CD56⁺CD8⁺ T (p = 0.02) and a decreased frequency of NKG2D⁺CD8⁺ T (p = 0.02) compared with healthy donors. Unlike CD8⁺ T cells from healthy donors, CD8⁺ T cells from TB patients exhibit greater cytotoxicity, mediated by HLA class I molecules, on autologous monocytes in the presence of mycobacterial antigens (p = 0.005). Finally, TB patients have a proinflammatory profile characterized by serum high level of TNF‐α (p = 0.02) and IL‐8 (p = 0.0001), but, interestingly, IL‐4 (p = 0.002) was also increased compared with healthy donors. Our data show evidence regarding the highly cytotoxic status of CD8⁺ T cells in Mtb infection. These cytotoxic cells restricted to HLA‐A, B, and C could be used to optimize strategies for designing new TB vaccines or for identifying markers of disease progression.     

The immune system utilizes two distinct effector mechanisms of T cells depending on two different life cycle stages of a single pathogen,Toxoplasma gondii,to control its cerebral infection

Toxoplasma gondii takes two different life cycle stages within intermediate hosts including humans. Tachyzoites proliferate during the acute stage, and they transform into cysts to establish a chronic infection preferentially in the brain. IFN-γ production by infiltrated CD4⁺ and CD8⁺ T cells is required for the prevention of cerebral tachyzoite growth. IFN-γ production by brain-resident cells, most likely microglia, plays a key first line defense role to facilitate both innate and T cell-mediated protective immunity to control the tachyzoite growth. IFN-γ produced by brain-resident cells activates cerebral expression of IFN-dependent effector molecules to suppress tachyzoite growth during the early stage of infection. Their IFN-γ production also induces an expression of CXCL9 and CXCL10 chemokines to recruit immune T cells into the brain, and upregulates cerebral expression of MHC class I and II molecules for antigen presentation to the recruited T cells to activate their IFN-γ production. CD8⁺ T cells also have the activity to remove T. gondii cysts from the brains of infected hosts. Of interest, the anti-cyst activity of CD8⁺ T cells does not require their IFN-γ but does require perforin. Notably, we discovered that CD8⁺ cytotoxic T cells penetrate in the cysts in a perforin-mediated manner, which induces morphological deterioration and destruction of the cysts and an accumulation of microglia and macrophages for their elimination. Thus, the immune system employs two distinct effector mechanisms mediated by IFN-γ or perforin depending on two different life cycle stages of a single pathogen, T. gondii, to control its cerebral infection.     

A combination trial of vaccine plus ipilimumab in metastatic castration-resistant prostate cancer patients: immune correlates

We recently reported the clinical results of a Phase I trial combining ipilimumab with a vaccine containing transgenes for prostate-specific antigen (PSA) and for a triad of costimulatory molecules (PROSTVAC) in patients with metastatic castration-resistant prostate cancer. Thirty patients were treated with escalating ipilimumab and a fixed dose of vaccine. Of 24 chemotherapy-naïve patients, 58 % had a PSA decline. Combination therapy did not exacerbate the immune-related adverse events associated with ipilimumab. Here, we present updated survival data and an evaluation of 36 immune cell subsets pre- and post-therapy. Peripheral blood mononuclear cells were collected before therapy, at 13 days and at 70 days post-initiation of therapy, and phenotyped by flow cytometry for the subsets of T cells, regulatory T cells, natural killer cells, and myeloid-derived suppressor cells. Associations between overall survival (OS) and immune cell subsets prior to treatment, and the change in a given immune cell subset 70 days post-initiation of therapy, were evaluated. The median OS was 2.63 years (1.77–3.45). There were trends toward associations for longer OS and certain immune cell subsets before immunotherapy: lower PD-1⁺Tim-3^NEGCD4_EM (P = 0.005, adjusted P = 0.010), higher PD-1^NEGTim-3⁺CD8 (P = 0.002, adjusted P = 0.004), and a higher number of CTLA-4^NEG Tregs (P = 0.005, adjusted P = 0.010). We also found that an increase in Tim-3⁺ natural killer cells post- versus pre-vaccination associated with longer OS (P = 0.0074, adjusted P = 0.015). These results should be considered as hypothesis generating and should be further evaluated in larger immunotherapy trials.     

Immunologic hierarchy,class II MHC promiscuity,and epitope spreading of a melanoma helper peptide vaccine

Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4⁺ T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8⁺ T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund’s adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4⁺ and CD8⁺ T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4⁺ T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3_281–295 (49 %) and tyrosinase_386–406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8⁺ T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4⁺ and CD8⁺ T cell responses against melanoma antigens. CD4⁺ T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8⁺ T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.