scholarly journals 195 Stimulation of tumor infiltrating B-cells improves ex-vivo TIL expansion for melanoma immunotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A206-A206
Author(s):  
Renata Rossetti ◽  
Leticia Tordesillas ◽  
Matthew Beatty ◽  
Yian Ann Chen ◽  
Dongliang Du ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Tumor Infiltrating Lymphocytes ◽  
Effector Memory ◽  
Cancer Center ◽  
High Dose ◽  
Standard Protocol ◽  
Melanoma Tumor

BackgroundThe immunogenic nature of melanoma has been exploited for the development of adoptive transfer of ex-vivo expanded tumor infiltrating lymphocytes (TIL). This adoptive cell transfer therapy has overall response rates of around 50%. Multiple factors may determine the quality of the TIL product including components of the tumor microenvironment. B-cells are frequently found in melanoma metastasis, and display signs of antigen experience. Recently, B-cell tumor infiltration has been associated with improved clinical responses to immune checkpoint inhibitors,1 2 but their role in TIL therapy remains unexplored. Considering the potential role of B cells, we aim to develop strategies to enhance the quality of TIL products through B-cell stimulation during ex-vivo TIL expansion.MethodsWe stimulated melanoma infiltrating B-cells using human recombinant CD40L on the first day of ex-vivo TIL expansion. Thirteen samples were expanded from melanoma tumor single cell suspensions, in high dose IL-2 alone (standard protocol), or in high dose IL-2 plus CD40L. After up to four weeks of expansion, the TIL phenotype was analyzed by flow cytometry.ResultsThe expansion success rate from the frozen tumor digests was 69% (95% CI: 38.6–90.9%) in the CD40L treatment condition compared to 23% with the standard protocol. Also, TILs cultured in the presence of CD40L expanded to higher numbers than with the standard protocol (P = 0.02). Interestingly, most of the samples expanded with CD40L had a significant increase in the percentage of CD4+ T cells (P = 0.03), but not to the detriment of the absolute number of CD8+ T cells. Treatment with CD40L increased the percentage of effector memory-like T cells (P = 0.03) and of CD39- CD69- T cells (P < 0.05), which were recently associated with response to TIL therapy.3ConclusionsThis preliminary work demonstrates that the stimulation with CD40L at the initiation of TIL culture leads to enhanced TIL expansion and an increase in CD4+ T cells with an effector memory-like and stem-like phenotype. Our group and others have previously described cases of patients who had tumor regression after receiving TIL therapy that were predominantly CD4+ T cells, suggesting that expansion of the CD4+ TIL repertoire may enhance TIL therapy.4AcknowledgementsThis work has been supported in part by the Flow Cytometry, Genomics and Biostatistics and Bioinformatics Core Facilities at Moffitt Cancer Center, an NCI designated Comprehensive Cancer Center (P30-CA076292). We acknowledge Moffitt’s Melanoma Center of Excellence for the financial support.ReferencesCabrita R, Lauss M, Sanna A. Tertiary lymphoid structures improve immunotherapy and survival in melanoma. Nature 2020;577:561–565.Petitprez F, de Reynies A, Keung EZ. B cells are associated with survival and immunotherapy response in sarcoma. Nature 2020;577:556–560.Krishna S, Lowery FJ, Copeland AR. Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer. Science 2020;370:1328–1334.Friedman KM, Prieto PA, Devillier LE. Tumor-specific CD4+ melanoma tumor-infiltrating lymphocytes. J Immunother 2012;35:400–408.Ethics ApprovalThe study was approved by Advarra IRB, approval number MCC20559.

scholarly journals Dual T cell– and B cell–intrinsic deficiency in humans with biallelic RLTPR mutations

2016 ◽  
Vol 213 (11) ◽  
pp. 2413-2435 ◽  
Author(s):  
Yi Wang ◽  
Cindy S. Ma ◽  
Yun Ling ◽  
Aziz Bousfiha ◽  
Yildiz Camcioglu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Cell Phenotype ◽  
Loss Of Function ◽  
Inborn Errors

Combined immunodeficiency (CID) refers to inborn errors of human T cells that also affect B cells because of the T cell deficit or an additional B cell–intrinsic deficit. In this study, we report six patients from three unrelated families with biallelic loss-of-function mutations in RLTPR, the mouse orthologue of which is essential for CD28 signaling. The patients have cutaneous and pulmonary allergy, as well as a variety of bacterial and fungal infectious diseases, including invasive tuberculosis and mucocutaneous candidiasis. Proportions of circulating regulatory T cells and memory CD4+ T cells are reduced. Their CD4+ T cells do not respond to CD28 stimulation. Their CD4+ T cells exhibit a "Th2" cell bias ex vivo and when cultured in vitro, contrasting with the paucity of "Th1," "Th17," and T follicular helper cells. The patients also display few memory B cells and poor antibody responses. This B cell phenotype does not result solely from the T cell deficiency, as the patients’ B cells fail to activate NF-κB upon B cell receptor (BCR) stimulation. Human RLTPR deficiency is a CID affecting at least the CD28-responsive pathway in T cells and the BCR-responsive pathway in B cells.

Ex Vivo Fludarabine Selectively Depletes Human Naive T-Cell Subsets: A Step Toward Modifying Donor Lymphocyte Infusion.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1071-1071
Author(s):  
Melody M. Smith ◽  
Cynthia R. Giver ◽  
Edmund K. Waller ◽  
Christopher R. Flowers
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Memory T Cells ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Naïve T Cell ◽  
Naive T Cell

Abstract Ex vivo modification of donor lymphocytes with purine analogs (mDL) may help to minimize graft versus host disease (GvHD) while providing beneficial graft versus leukemia (GvL) effects. In a murine model system, we have shown that allogeneic donor splenocytes, treated with fludarabine ex vivo have significantly reduced GvHD activity when transferred to irradiated recipient mice, and retain anti-viral and GvL activities (Giver, 2003). This effect appears to be mediated by relative depletion of donor CD4 CD44low, “naive” T-cells. As a first step toward developing mDL for use in patients, we sought to evaluate the effects of ex vivo fludarabine exposure on human T-cell subsets, and to determine the minimum dose of fludarabine required to achieve this effect. Methods: Peripheral blood mononuclear cell samples from 6 healthy volunteers were evaluated at 0, 24, 48, and 72 hour time points after ex vivo incubation in varying dosages of fludarabine: 2, 5, and 10(n=3) mcg/ml. Fludarabine incubated samples were compared to samples that received no fludarabine (untreated). The total viable cell number was determined and the fractions and absolute numbers of viable CD4 and CD8 naïve and memory T-cells were determined using flow cytometry after incubation with 7-AAD (dead cell stain), CD4, CD8, CD45RA, CD62L, and CCR7 antibodies, and measuring the total viable cells/ml. Results: The numbers of viable CD4 and CD8 T-cells remained relatively stable in control cultures. Without fludarabine, the average viability at 72 hr of naive and memory T-cells were 92% and 77% for CD4 and 86% and 63% for CD 8 (Fig. 1A). Naive CD4 T-cells were more sensitive to fludarabine-induced death than memory CD4 cells. At 72 hr, the average viability of fludarabine-treated naive CD4 T-cells was 33% at 2 mcg/ml (8.2X the reduction observed in untreated cells) and 30% at 5 mcg/ml, while memory CD4 T-cells averaged 47% viability at 2 mcg/ml (2.3X the reduction observed in untreated cells) (Fig. 1B) and 38% at 5 mcg/ml. The average viability of naive CD8 T-cells at 72 hr was 27% at 2 mcg/ml and 20% at 5 mcg/ml, while memory CD8 T-cell viability was 22% at 2 mcg/ml and 17% at 5 mcg/ml. Analyses on central memory, effector memory, and Temra T-cells, and B-cell and dendritic cell subsets are ongoing. The 5 and 10 mcg/ml doses also yielded similar results in 3 initial subjects, suggesting that 2 mcg/ml or a lower dose of fludarabine is sufficient to achieve relative depletion of the naive T-cell subset. Conclusions: Future work will determine the minimal dose of fludarabine to achieve this effect, test the feasibility of using ex vivo nucleoside analog incubation to reduce alloreactivity in samples from patient/donor pairs, and determine the maximum tolerated dose of mDL in a phase 1 clinical trial with patients at high risk for relapse and infectious complications following allogeneic transplantation. Figure Figure

Increased Expression of PD-1 on CD4+ T Cells from Patients with Chronic Lymphocytic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4154-4154
Author(s):  
Mary M Sartor ◽  
David J Gottlieb
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Nk Cells ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Lymphocytic Leukemia ◽  
Effector Memory ◽  
Cd4 Cells ◽  
Normal Peripheral Blood

Abstract Although the predominant finding in patients with chronic lymphocytic leukemia (CLL) is an expansion of monoclonal B lymphocytes, a polyclonal expansion of T cells co-exists in CLL patients. Allogenic stem cell transplants for CLL suggest that a significant graft versus leukaemia effect mediated through recognition of minor MHC or leukaemia specific antigens can be achieved. Since it appears that the immune system and probably T cells recognise CLL cells, it is possible that one or more T cell defects might contribute to the initiation or maintenance of a clone of CLL lymphocytes. PD-1 is a coinhibitory molecule that is expressed on T cells in patients with chronic viral infections. It has been suggested that PD-1 expression might be a marker of cell exhaustion due to antigenic overstimulation. We examined the expression of PD-1 and its naturally occurring ligands PD-L1 and PD-L2 on both B and T cells in patients with CLL and compared this with expression on normal peripheral blood mononuclear cells. We found that PD-1 was expressed on over 10% of CD4+ T cells in 7 of 9 cases of CLL (mean 22±16%) but not on CD4+ T cells in any of 9 normal donors (mean 0±0%), p=0.0009. There was no difference in PD-1 expression on CD8+ or CD14+ PBMCs from CLL patients and normal donors (for CD8+ 24±21% and 19±16% for CLL and normals; for CD14+ 58±16% and 71±31% for CLL and normals). More than 10% of CD5+/19+ CLL cells expressed PD-1 in 7 of 10 cases (mean 18±18%) while more than 10% of normal B cells from 6 of 7 donors also expressed PD-1 (mean 49±30%). We examined the expression of PD-1 on naïve, central memory, effector memory and terminally differentiated subsets of CD4+ cells (CD62L+CD45RA+, CD62L+CD45RA−, CD62L−CD45RA− and CD62L−CD45RA+ respectively) from CLL patients and normal donors. The expression of PD-1 was higher on CD4+ cells from CLL patients in all subsets. The effect was most prominent in the effector memory subset (mean 54±4% for CLL patients versus 26±17% for normal donors, p=0.02). We looked for expression of PD-L1 and PD-L2 on T cells, B cells, monocytes and NK cells from CLL patients and normal donors. PD-L1 was only expressed on monocytes (mean 30±23%) and NK cells (mean 14±19%) from CLL patients and on monocytes from normal donors (mean 35±26%). There was no expression of PD-L2 on any cell type in either CLL patients or normal donors. We conclude that there is increased expression of the co-inhibitory molecule PD-1 on CD4+ T cells in patients with CLL. Ligation of PD-1 by PD-L1 expressed on monocytes or NK cells could inhibit immune responses to tumor and infectious antigens leading to persistence of clonally expanded cells and predisposition to opportunistic pathogens.

Anti-GvHD Effect of Antithymocyte Globulin Is Mediated by Antibodies Binding to T Cells and Regulatory NK Cells, and Less So or Not at All by Antibodies Binding to Other Mononuclear Cell Subsets

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2346-2346
Author(s):  
Mette Hoegh-Petersen ◽  
Minaa Amin ◽  
Yiping Liu ◽  
Alejandra Ugarte-Torres ◽  
Tyler S Williamson ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Antithymocyte Globulin ◽  
Chronic Gvhd ◽  
Wilcoxon Test ◽  
Effector Memory

Abstract Abstract 2346 Introduction: Polyclonal rabbit-anti-human T cell globulin may decrease the likelihood of graft-vs-host disease (GVHD) without increasing the likelihood of relapse. We have recently shown that high levels of antithymocyte globulin (ATG) capable of binding to total lymphocytes are associated with a low likelihood of acute GVHD grade 2–4 (aGVHD) as well as chronic GVHD needing systemic therapy (cGVHD) but not increased likelihood of relapse (Podgorny PJ et al, BBMT 16:915, 2010). ATG is polyclonal, composed of antibodies for antigens expressed on multiple cell subsets, including T cells, B cells, NK cells, monocytes and dendritic cells. These cell subsets may play a role in the pathogenesis of GVHD. The anti-GVHD effect of ATG may be mediated through killing/inhibition of one or several of these cell subsets (eg, T cells) or their subsets (eg, naïve T cells as based on mouse experiments naïve T cells are thought to play a major role in the pathogenesis of GVHD). To better understand the mechanism of action of ATG on GVHD, we set out to determine levels of which ATG fraction (capable of binding to which cell subset) are associated with subsequent development of GVHD. Patients and Methods: A total of 121 patients were studied, whose myeloablative conditioning included 4.5 mg/kg ATG (Thymoglobulin). Serum was collected on day 7. Using flow cytometry, levels of the following ATG fractions were determined: capable of binding to 1. naïve B cells, 2. memory B cells, 3. naïve CD4 T cells, 4. central memory (CM) CD4 T cells, 5. effector memory (EM) CD4 T cells, 6. naïve CD8 T cells, 7. CM CD8 T cells, 8. EM CD8 T cells not expressing CD45RA (EMRA-), 9. EM CD8 T cells expressing CD45RA (EMRA+), 10. cytolytic (CD16+CD56+) NK cells, 11. regulatory (CD16-CD56high) NK cells, 12. CD16+CD56− NK cells, 13. monocytes and 14. dendritic cells/dendritic cell precursors (DCs). For each ATG fraction, levels in patients with versus without aGVHD or cGVHD were compared using Mann-Whitney-Wilcoxon test. For each fraction for which the levels appeared to be significantly different (p<0.05), we determined whether patients with high fraction level had a significantly lower likelihood of aGVHD or cGVHD than patients with low fraction level (high/low cutoff level was determined from ROC curve, using the point with maximum sum of sensitivity and specificity). This was done using log-binomial regression models, ie, multivariate analysis adjusting for recipient age (continuous), stem cell source (marrow or cord blood versus blood stem cells), donor type (HLA-matched sibling versus other), donor/recipient sex (M/M versus other) and days of follow up (continuous). Results: In univariate analyses, patients developing aGVHD had significantly lower levels of the following ATG fractions: binding to naïve CD4 T cells, EM CD4 T cells, naïve CD8 T cells and regulatory NK cells. Patients developing cGVHD had significantly lower levels of the following ATG fractions: capable of binding to naïve CD4 T cells, CM CD4 T cells, EM CD4 T cells, naïve CD8 T cells and regulatory NK cells. Patients who did vs did not develop relapse had similar levels of all ATG fractions. In multivariate analyses, high levels of the following ATG fractions were significantly associated with a low likelihood of aGVHD: capable of binding to naïve CD4 T cells (relative risk=.33, p=.001), EM CD4 T cells (RR=.30, p<.001), naïve CD8 T cells (RR=.33, p=.002) and regulatory NK cells (RR=.36, p=.001). High levels of the following ATG fractions were significantly associated with a low likelihood of cGVHD: capable of binding to naïve CD4 T cells (RR=.59, p=.028), CM CD4 T cells (RR=.49, p=.009), EM CD4 T cells (RR=.51, p=.006), naïve CD8 T cells (RR=.46, p=.005) and regulatory NK cells (RR=.55, p=.036). Conclusion: For both aGVHD and cGVHD, the anti-GVHD effect with relapse-neutral effect of ATG appears to be mediated by antibodies to antigens expressed on naïve T cells (both CD4 and CD8), EM CD4 T cells and regulatory NK cells, and to a lesser degree or not at all by antibodies binding to antigens expressed on B cells, cytolytic NK cells, monocytes or DCs. This is the first step towards identifying the antibody(ies) within ATG important for the anti-GVHD effect without impacting relapse. If such antibody(ies) is (are) found in the future, it should be explored whether such antibody(ies) alone or ATG enriched for such antibody(ies) could further decrease GVHD without impacting relapse. Disclosures: No relevant conflicts of interest to declare.

In Vitro Co-Culture of CLL-B Cells Reveals Long-Term Survival, Proliferation, and Maintenance of Telomere Length

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 350-350
Author(s):  
Ceri H Jones ◽  
Thet Thet Lin ◽  
Elisabeth Jane Walsby ◽  
Guy E Pratt ◽  
Christopher Fegan ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Telomere Length ◽  
Erosion Rate ◽  
Ex Vivo ◽  
Effector Memory ◽  
Long Term Culture

Abstract Telomere length is a prognostic factor in Chronic Lymphocytic Leukemia (CLL) with short telomere length a powerful predictor of early time to first treatment and reduced overall survival. However, little is known about telomere dynamics through the course of an individual patient's disease. Our recent longitudinal analysis of CLL B-cell telomere length revealed very little dynamic change within individual patients with a mean erosion rate of -52bp/year (p=0.05). In marked contrast, T-cells derived from the same patients showed a significantly higher mean erosion rate of -119bp/year (p=0.02) with a median follow up time of 69 months. Here we present data derived from long-term in-vitro co-culture of peripheral blood from CLL patients coupled with temporal analysis of their telomere length dynamics. We utilized a multi-cellular co-culture system, comprised of autologous T-cells and CD40L-expressing mouse fibroblasts, to maintain CLL cells in long-term culture. Patient-derived peripheral blood mononuclear cells (n=16) were maintained for a median of 70 days (range 54-154); samples were analyzed every two weeks for tumor cell telomere length and evidence of proliferation. We used fluorescence-activated cell sorting (FACS) to sort populations of CD19+CD5+ CLL B-cells and CD3+ T-cells from each of the cultures. We then performed high-resolution single telomere length analysis (STELA) on these sorted subsets of cells and analyzed their telomere dynamics over this extended time course. Analysis of CLL B-cells from these cultures revealed significantly increased Ki-67+ at day 14 when compared to day 0 (p<0.001) and this was evident for the duration of the cultures. Despite sustained tumor cell proliferation, we observed no significant difference in the CLL B-cell telomere length with a mean TL at the start of 4.5kb vs 4.3kb at the end (p=0.14). The presence of T-cells was shown to be critical for the maintenance of the long-term cultures in two ways. Firstly, cultures that were treated with 4μM fludarabine showed a catastrophic reduction in T-cells (p=0.01), which was associated with a significantly shorter duration of survival of CLL B-cells when compared to untreated controls (median 17.5 days (range 7-70); p<0.001). Secondly, it proved impossible to maintain T-cell depleted, purified CLL B-cells, in long-term culture. T-cells isolated from the long-term cultures showed evidence of proliferation with Ki-67+ again being increased at day 14 in comparison to baseline (p=0.003). Furthermore, T-cells derived from these cultures showed a significant alteration in subset composition over time with a decrease in the numbers of naive CD4+ (p=0.05) and CD8+ (p=0.02) T-cells and a corresponding increase in effector memory (p=0.2) and terminally differentiated effector memory (EMRA) subsets (p=0.07). In conclusion, this study demonstrates that we have developed a robust, long-term culture method for the maintenance of CLL cells. Despite evidence of sustained CLL proliferation, CLL B-cells showed little telomere length erosion during long-term co-culture and this is compatible with our recent ex-vivo analysis, which showed that the telomere length of CLL B-cells are remarkably stable with a mean erosion rate of only -52bp/year. In both ex-vivo and in-vitro analysis, telomere erosion correlated with starting telomere length (r2=0.14, p=0.04 and r2=0.3 p=0.03 respectively). Taken together, our in-vitro and ex-vivo data imply that the radically short telomeres observed in some CLL patients are not the result of increased proliferation of the malignant B-cell, but rather the mutagenic event occurs in a B-cell which already has short telomeres. Furthermore, our novel long-term culture model has reinforced the vital role of T-cells in sustaining CLL B-cells viability and proliferation in-vitro. Given the consistent skewing of the T-cell pool towards a memory phenotype it seems unlikely that this is driven in-vitro by cognate TCR antigen recognition but rather a cytokine-mediated response. Disclosures Fegan: Gilead Sciences: Honoraria; Roche: Honoraria; AbbVie: Honoraria.

scholarly journals Optimizing CD4+ T-Cells Transduction Protocol for Gene Therapy of HIV-1 Infected Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4429-4429
Author(s):  
Amani Ouedrani ◽  
Lounes Djerroudi ◽  
Isabelle Hmitou ◽  
Marina Cavazzana ◽  
Fabien Touzot
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Cd4 T Cells ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Effector Memory ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Hiv 1

Abstract Gene therapy represents an alternative and promising strategy that could provide a path to a curative therapy for HIV-1 infection. One approach involves the introduction of protective gene into a cell, thereby conferring protection against HIV. We plan to conduct an open label phase I/II gene therapy trial for HIV-1 infected patients presenting with lymphoma. The patients will received autologous hematopoietic stem cells transplantation with gene modified CD34+ cells and CD4+ T-cells. CD34+ and CD4+ will be ex vivo transduced by the LVsh5/C46 lentiviral vector (Cal-1, Calimmune, Inc. Tucson, USA). LVsh5/C46 is a SIN lentiviral vector that inhibits two crucial steps of CD4+ T cell infection by the HIV virus: (i) attachment of the virus to its target by downregulation of CCR5 via a short hairpin RNA, (ii) fusion of the virus to the target cell through expression of the C46 inhibitor. We developed a transduction process for CD4+ T-cells using the TransAct™ reagent (Miltenyi Biotec, Bergisch Gladbach , Germany) for CD4+ T-cells activation. Compared to previously published T-cells transduction protocols, the use of Miltenyi TransAct™ permits an equivalent efficacy of transduction - evaluated by measurement of vector copy number through quantitative PCR - without major phenotypic modification. Indeed, CD4+ T-cells ex vivo transduced after activation with the TransAct™ reagent display very few changes in their surface marker with conservation of naive (CCR7+CD62L+CD45RA+), central memory (CCR7+CD62L+CD45RA-) and effector memory (CCR7-CD62L-CD45RA-) subsets in superimposable proportions as initially. Moreover, expression of CD25 remains below 15-25% of cells suggesting a more "gentle " activation of the transduced CD4+ T-cells. Our transduction process had no significant impact in TCRβ repertoire diversity as evaluated by high-throughput sequencing and analyzis of diversity through the Gini-Simpson index or the Shannon index. Finally, transduced CD4 + T-cells retained the ability to to be primed towards the TH1, TH2 and TH17 pathways suggesting that the transduction protocol used did not alter the functional properties of the target cells. Disclosures No relevant conflicts of interest to declare.

P16.06 Immunophenotyping of tumor-infiltrating T cells in primary CNS lymphoma

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii57-ii57
Author(s):  
S Schliffke ◽  
C Maire ◽  
M Holz ◽  
C Bokemeyer ◽  
M Westphal ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Primary Cns Lymphoma ◽  
Great Promise ◽  
Cns Lymphoma ◽  
High Dose ◽  
Dismal Prognosis

Abstract BACKGROUND Primary CNS lymphoma represents a malignant disease with dismal prognosis. Standard of care is high dose chemotherapy and radiation. However, this combination cannot be applied to the elderly and fragile population. Immunotherapy holds great promise to be effective in these patients. This study therefore aims to explore the phenotype of tumor-infiltrating lymphocytes (TIL) in order to analyze the potential for immune checkpoint inhibition. MATERIAL AND METHODS We performed ex vivo multicolor flow-cytometry on surgical specimens of nine patients with intracerebral lymphoma, including seven with primary CNS lymphoma after isolation of TILs following standard protocols. Data was analyzed using a Fortessa LSR flow cytometer and Diva software. The study was approved by the local ethics committee (PV4904). RESULTS Our ex vivo phenotyping demonstrated a predominant infiltration of CD8+ T cells, which outnumber CD4+ T cells by a ratio of 2:1 (p&lt;0.01). Regulatory T cells (Tregs) were not increased in the tumor microenvironment and the NK cell frequency was reduced compared to the peripheral blood. While CD4+ T helper cells displayed significantly increased surface expression of multiple activation and checkpoint markers, including TIGIT, PD-1, Tim3 and CD57, cytotoxic CD8+ T cells predominantly expressed only TIGIT and PD-1. On average 70% and 80% of CD8+ T cells expressed PD-1 and TIGIT, respectively, compared to 35% and 60% of PD-1 and TIGIT on CD4+ T cells (p&lt;0.05). CD8+ T cells furthermore showed an increased expression of CD39 and a simultaneous downregulation of CD73, both ectoenzyms involved in the modulation of intratumoral ATP, thereby indicating a metabolic immune modulation by the tumor. CONCLUSION Taken together, our study demonstrates a strong infiltration of cytotoxic CD8+ T cells into cerebral lymphoma, which potentially can be disinhibited using checkpoint immunotherapy. Our profiling suggests that PD-1 and TIGIT present appealing targets for such kind of immune disinhibition.

Use of a Potassium Channel Blocker to Inhibit Effector-Memory T Cells Isolated from Recipients of Allogeneic BMSCT.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1154-1154
Author(s):  
Cheryl Cameron ◽  
Jeffrey Woodliff ◽  
Laura McOlash ◽  
Parameswaran Hari ◽  
Christopher Bredeson ◽  
...  
Keyword(s):  
T Cells ◽  
Potassium Channel ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Chronic Gvhd ◽  
Specific Inhibitor ◽  
Central Memory ◽  
Effector Memory ◽  
Channel Blockers

Abstract Following allogeneic blood or marrow stem cell transplantation (BMSCT), donor T cells are persistently exposed to host alloantigens and develop into effector-memory (TEM) cells. CD4+ T-EM cells are thought to play a role in perpetuating chronic GVHD (cGVHD) (K. Yamashita et al., BLOOD103:3986, 2004). In contrast to central memory (T-CM) and naïve CD4+ T cells, human CD4+ T-EM cells over-express the voltagegated potassium channel Kv1.3 when activated. As a result, Kv1.3 has emerged as an attractive pharmacological target for the selective suppression of T-EM cells, especially in the setting of autoimmune disease (C. Beeton et al., Mol Pharm67:1369, 2005). We hypothesized that over-expression of Kv1.3 by T-EM cells obtained from allogeneic BMSCT patients with cGVHD would make them preferential targets for Kv1.3-specific potassium channel blockers. To our knowledge, no one has examined the effect of Kv1.3 blockers on T-EM cells from patients with cGVHD. In this study, we isolated T-EM cells as well as central-memory (T-CM) and naïve T cells from fresh (n = 5) and frozen (n = 4) peripheral blood (PB) of patients who had undergone allogeneic BMSCT more than 6 months earlier as well as from normal volunteers by fluorescence-activated cell sorting (FACS). All BMSCT patients had some clinical evidence of cGVHD. T-EM cells were identified by their characteristic immunophenotype: CD4+ CD45RO+/RA- CCR7- CD62L Low. The percentage of CD4+ T-EM cells in 9 patients with cGVHD was 45 ± 21% (range 19%–70%) as compared to approximately 11% in healthy controls. Sorted T-EM, T-CM and naïve CD4+ T cells were polyclonally activated ex vivo using Dynal beads coupled with anti-CD3/CD28 monoclonal antibodies. The proportion of cells secreting gamma interferon (γIFN) was quantified using EliSpot assays to assess functional activity. We first compared the kinetics of γIFN secretion by activated T-EM cells to that of T-CM and naïve T-cells. Only T-EM cells secreted γIFN within 24 hrs of activation; T-CM cells secreted γIFN ≥ 48 hrs after activation, and naïve T cells required ≥ 96 hrs. ShK peptide, a highly specific inhibitor of Kv1.3 channels derived from the sea anemone Stichodactyla helianthus, was added at the initiation of 24-hr cultures. Dose response assays confirmed that T-EM cells in both BMSCT patients and normals were preferential targets of the Kv1.3-specific peptide. On average, T-EM cells were inhibited 26%, 55%, and 69% with ShK concentrations of 1, 10 and 100 nM, respectively. With 100nM ShK, T-EM cells sorted from the peripheral blood of BMSCT patients were inhibited an average of 44 ± 21% (range 17–67%). Our data, using polyclonally activated T-EM cells, support further testing of Kv1.3 blockers against host-alloantigen-specific T-EM cells obtained from BMSCT recipients and as a potential therapeutic agent for cGVHD.

scholarly journals Broad phenotypic alterations and potential dysfunction of lymphocytes in individuals clinically recovered from COVID-19

2021 ◽  
Author(s):  
Jingyi Yang ◽  
Maohua Zhong ◽  
Ejuan Zhang ◽  
Ke Hong ◽  
Qingyu Yang ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Cell Subset ◽  
Effector Memory ◽  
Peripheral Blood Mononuclear ◽  
Follicular Helper ◽  
Hla Dr ◽  
Ifn Γ

Abstract Although millions of patients have clinically recovered from COVID-19, little is known about the immune status of lymphocytes in these individuals. In this study, the peripheral blood mononuclear cells (PBMCs) of a clinically recovered (CR) cohort were comparatively analyzed with those of an age- and sex-matched healthy donor (HD) cohort. We found that CD8+ T cells in the CR cohort had higher numbers of effector T cells and effector memory T cells but lower Tc1 (IFN-γ+), Tc2 (IL-4+), and Tc17 (IL-17A+) cell frequencies. The CD4+ T cells of the CR cohort were decreased in frequency, especially the central memory T cell subset. Moreover, CD4+ T cells in the CR cohort showed lower PD-1 expression and had lower frequencies of Th1 (IFN-γ+), Th2 (IL-4+), Th17 (IL-17A+), and circulating follicular helper T (CXCR5+PD-1+) cells. Accordingly, the proportion of isotype-switched memory B cells (IgM−CD20hi) among B cells in the CR cohort showed a significantly lower proportion, although the level of the activation marker CD71 was elevated. For CD3−HLA-DR− lymphocytes in the CR cohort, in addition to lower levels of IFN-γ, granzyme B, and T-bet, the correlation between T-bet and IFN-γ was not observed. Additionally, by taking into account the number of days after discharge, all the phenotypes associated with reduced function did not show a tendency toward recovery within 4‒11 weeks. The remarkable phenotypic alterations in lymphocytes in the CR cohort suggest that SARS-CoV-2 infection profoundly affects lymphocytes and potentially results in dysfunction even after clinical recovery.

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