In Vitro Generation of Multi-Epitope Specific CD4+ T Helper Cells for Adoptive Immunotherapy of Leukemia

Abstract Introduction: Acute myeloid leukemia (AML) and other hematological malignancies that constitutively express MHC class II molecules represent the ideal target for leukemia-specific CD4+ T helper (Th) cells. CD4+ Th cells are central to the functioning of the immune system. They regulate adaptive immunity against infections and drive pathogenic responses in many autoimmune diseases. Our preclinical studies indicate that adoptively transferred antigen specific Th cells efficiently eradicate even advanced tumors in mice, showing functional advantage over their better characterized classical CD8+ cytotoxic counterparts. Unfortunately, the production of human class II-restricted Th cells is complex and challenging. Consequently, the clinical activity of tumor-specific Th cells has not been systematically evaluated and very little is known about their therapeutic potential in humans. Striking evidence of their enormous power comes from some anecdotal clinical reports of complete regression of metastatic cancers upon transfer of antigen-specific Th cells. Methods and Results: We investigated a reliable GMP-compatible method for in vitro expansion of antigen-specific CD4+ T cells targeting common leukemia associated antigens (LAAs) Willm's Tumor antigen 1 (WT1) and Preferentially Expressed Antigen in Melanoma (PRAME) for future adoptive immunotherapy in the setting of allogeneic stem cell transplantation (SCT). We hypothesized that the naïve CD4+ T cell compartment, rather than the bulk Th cell population could be a superior source for generating a tumor antigen specific cell product. To test this hypothesis bulk and naïve CD4+ T cells were isolated from the peripheral blood of normal donors by magnetic bead separation. Purified T cells were stimulated in vitro with autologous dendritic cells (DCs) pulsed with overlapping 15 amino-acid long peptide (pepmixes) spanning the length of both proteins. Two rounds of stimulation were performed in presence of IL-7, IL-15 and later +/- addition of IL-1, IL-6 and IL-23. IL-2 in low concentration was supplemented during the second round of stimulation. At the end of the second expansion the cells were tested for reactivity by intracellular cytokine production using flow cytometry (FACS) upon stimulation with cognate LAAs or irrelevant control pepmixes. In T cell cultures derived from naïve CD4+ T cells we observed robust induction of PRAME reactivity from majority of tested normal donors, while reactivity against WT1 was donor dependent. The frequency of LAAs in bulk CD4+ T cells was significantly lower in all cases, supporting the notion that pre-exiting memory Th cells have a competitive advantage over the LAA-specific precursor. T cell cultures supplemented with inflammatory cytokines demonstrated further enhancement of antigenic reactivity. Next we tested if LAA pepmix -stimulated T cells can recognize tumor targets. Naïve-derived PRAME and WT1 Th cells generated from normal SCT sibling donors produced IFN-γ, IL-2 and TNF-α upon exposure to fully HLA-matched AML blasts while no reactivity was seen in control CMV pp65-specific Th cells from the same donors. This observation suggests that LAA-specific CD4+ T cells induced with pepmixes have the ability to recognize physiologically-relevant tumor antigens. Conclusions: Here we report the feasibility of generating naïve-derived anti-leukemia CD4+ T cells from majority of normal donors. Removal of competing memory Th cells unmasks the LAA-specific reactivity, thus improving the reliability of the process. Importantly, these Th cells demonstrate highly-specific recognition of the tumor epitopes naturally processed by HLA-matched leukemic blasts, establishing the foundation for a future adoptive immunotherapy clinical trial in patients with hematological malignancy. Disclosures No relevant conflicts of interest to declare.

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Follicular Lymphoma Tumor Infiltrating T-Helper (Th) Cells Have the Same Intrinsic Polyfunctional Response Potential Upon Stimulation as Do Reactive and Normal Lymph Node Th Cells Despite Having Skewed Differentiation; Implications for Immunotherapy

Blood ◽

10.1182/blood.v116.21.3119.3119 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3119-3119

Author(s):

Shannon P. Hilchey ◽

Alexander F. Rosenberg ◽

Ollivier Hyrien ◽

Shelley Secor-Socha ◽

Matthew R. Cochran ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Lymph Nodes ◽

Follicular Lymphoma ◽

Cd4 T Cells ◽

Th2 Cells ◽

T Helper ◽

Th Cells ◽

Cytokine Profiles ◽

Th1 And Th2

Abstract Abstract 3119 Tumor infiltrating T-cells tend to be hypo-functional and this loss of function may be due to intrinsic T-cell defects, impaired antigen (Ag) presentation, and/or suppression induced by extrinsic components of the microenvironment, such as regulatory T-cells (Tregs). Each of these potential mechanisms has distinct implications on the potential efficacy of immunotherapy. To determine the functional potential of follicular lymphoma (FL) derived T-cells, we analyzed, by flow cytometry, T helper (Th) subsets and Staphylococcus enterotoxin B (SEB)-induced cytokine profiles of single cell suspensions from FL involved nodes (FL; n=8), reactive lymph nodes (RLN; n=7) and normal lymph nodes (NLN; n=6; obtained during vascular surgery). SEB was used as it directly triggers the T-cell receptor, abrogating the need for Ag presentation, and overcomes Treg mediated suppression. Herein we show that, relative to NLN, FL has decreased proportions of CD4+ T-cells having either a naïve (CD45RA+) or central memory (CD45RA−CCR7+) phenotype but increased proportions of effector memory T-cells (CD45RA−CCR7−). In addition, a higher percentage of pre-stimulation FL CD4+ T-cells show an activated (CD69+) phenotype as compared to that of RLN or NLN. Upon SEB stimulation, the FL CD4+ T-cells, like those from RLN and NLN, show an additional increase in the proportion of CD69+ cells, demonstrating that the FL derived CD4+ T-cells can be activated even further. We also show that upon stimulation with SEB; (a) the proportion of Th1 cells (IL-2+IFN-g+IL-4−) in FL is similar to that seen in RLN or NLN; (b) in contrast, we observe an increased frequency of primed uncommitted precursor Thpp cells (IL-2+IFN-g−IL-4−) in FL compared to that seen in either RLN or NLN; (c) an increased proportion of Th2 cells in FL compared with NLN and; (d) an increase in the proportion of Th17 cells in FL compared to that in RLN. Lastly, the proportions of FL Th cells producing 3 or 4 cytokines simultaneously, or poly-functional CD4+ T-cells, (PFT; PFT-3 producing IL-2, IFN-g and TNF-a or PFT-4 producing IL-2, IFN-g, TNF-a and MIP-1b), after SEB stimulation is similar to that seen in RLN or NLN. These data suggest that although there is skewed Th cell differentiation in FL, as compared to that of RLN or NLN, the intrinsic ability of the FL Th cells to elicit a clinically relevant effector response (both a Th1 and Th2 response) is fully preserved. In addition, the retention of effector function of FL Th cells is further supported by the fact that the proportions of these Th cells that have poly-functional cytokine profiles after SEB stimulation is similar in FL as compared to RLN or NLN. Indeed, poly-functionality of Th cells has been shown to correlate with the elicitation of protective immunity after vaccination for infectious diseases. Finally, the proportion of uncommitted Thpp cells after SEB stimulation is highest in FL. Thpp cells are non-polarized and can still differentiate into either Th1 or Th2 cells. They can also produce several chemokines and thus may play a role in shaping the FL microenvironment by recruiting other immune-effector cells as well as developing into Th1 and Th2 cells. Taken together, our data shows that FL Th cells are fully functional within the parameters of our assays, suggesting that these cells are intrinsically capable of mediating effective anti-tumor immune responses after immunotherapy. Therefore the hypo-functionality of FL T-cells is likely due to extrinsic factors which suppress T-cell function in vivo. Thus the challenge is to develop immunotherapeutic strategies that overcome these tumor associated extrinsic mechanisms, resulting in effective anti-tumor immunity. Disclosures: No relevant conflicts of interest to declare.

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Histone Deacetylase Inhibitors Induce Immuno-Dominant Suppression of Dendritic Cells.

Blood ◽

10.1182/blood.v106.11.456.456 ◽

2005 ◽

Vol 106 (11) ◽

pp. 456-456 ◽

Cited By ~ 1

Author(s):

Pavan Reddy ◽

Yoshinobu Maeda ◽

Raimon Duran-Struuck ◽

Oleg Krijanovski ◽

Charles Dinarello ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Hdac Inhibitors ◽

Class Ii ◽

Wild Type ◽

Tnf Α

Abstract We and others have recently demonstrated that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor with anti-neoplastic properties, reduces experimental acute graft-versus-host disease (GVHD). We have now investigated the mechanisms of action of two HDAC inhibitors, SAHA and ITF 2357, on allogeneic immune responses. Bone marrow derived dendritic cells (DCs) were preincubated with the HDAC inhibitors at nanomolar concentrations for 16–18 hours and stimulated with lipopolysaccharide (LPS). Pretreatment of DCs caused a significant reduction in the secretion of TNF-α, IL-12p70 and IL-6 compared to the untreated controls (P< 0.005). Similar effects were seen using human peripheral blood mononuclear cell derived DCs. Pre-treatment of both murine and human DCs also significantly reduced their in vitro stimulation of allogeneic T cells as measured by proliferation and IFN-γ production (P<0.01). We determined the in vivo relevance of these observations utilizing a mouse model where the responses of allogeneic donor bm12 T cells depended on the function of injected host B6 DCs would stimulate. Recipient Class-II −/− B6 (H-2b) received 11 Gy on day -1 and were injected with 4–5 x 106 wild type B6 DCs treated with SAHA or with media on days -1 and 0 and then transplanted with 2 x 106 T cells and 5 x 106 TCDBM cells from either syngeneic B6 or allogeneic bm12 donors. SAHA treatment of DCs significantly reduced expansion of allogeneic donor CD4+ T cells on day +7 after BMT compared to controls (P<0.05). SAHA treatment induced a similarly significant reduction in the expansion of CD8+ cells in Class I disparate [bm1→β2M−/−] model. In vitro, SAHA treatment significantly suppressed the expression of CD40 and CD80 but did not alter MHC class II expression. Surprisingly, when mixed with normal DCs at 1:1 ratio, SAHA treated DCs dominantly suppressed allogeneic T cell responses. The regulation of T cell proliferation was not reversible by addition of IL-12, TNF-α, IL-18, anti-IL-10 or anti-TGFβ, either alone or in combination. Suppression of allogeneic responses was contact dependent in trans-well experiments. To address whether the regulation of SAHA treated DCs required contact with T cells, we devised a three cell experiment where SAHA treated DCs lacked the capacity to present antigens to T cells. DCs from B6 MHC Class II deficient (H-2b) were treated with SAHA and co-cultured with wild type B6 (H-2b) DCs along with purified allogeneic BALB/c (H-2d) CD4+ T cells in an MLR. Allogeneic CD4+ T cells proliferated well, demonstrating the regulation to be dependent on contact between SAHA treated DCs and T cells. To address the in vivo relevance of this suppression, we utilized a well characterized [BALB/c →B6] mouse model of acute GVHD. Recipient B6 animals received 11Gy on day -1 and were injected with of 5 million host type SAHA treated or control DCs on days −1, 0, and +2. Mice were transplanted on day 0 with 2 x 106 T cells and 5 x 106 BM from either syngeneic B6 or allogeneic BALB/c donors. Injection of SAHA treated DCs resulted in significantly better survival (60% vs. 10%, P < 0.01) and significantly reduced serum levels of TNF-α, donor T cell expansion and histopathology of GVHD on day +7 after BMT compared to the controls. We conclue that HDAC inhibitors are novel immunomodulators that regulate DC function and might represent a novel strategy to prevent GVHD.

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Generation Of Anti-Tumor CD4+ T Helper Cells Using Genetically-Engineered Dendritic Cells Expressing Leukemia-Associated Antigens

Blood ◽

10.1182/blood.v122.21.2027.2027 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2027-2027

Author(s):

Dhanalakshmi Chinnasamy ◽

Pawel Muranski ◽

Manuel Franco-Colon ◽

Sawa Ito ◽

Nancy F. Hensel ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Mhc Class Ii ◽

Adoptive Immunotherapy ◽

Cd4 T Cells ◽

Conflicts Of Interest ◽

Class Ii ◽

High Avidity ◽

Th Cells ◽

Antigenic Targets

Abstract Adoptive transfer of antigen-specific T cells is a potentially curative strategy for patients with solid tumors and leukemia. Most clinical trials of adoptive T cell therapy have used cytotoxic CD8+ T cells recognizing MHC class I-restricted tumor antigens. Despite overwhelming evidence suggesting the fundamental influence of CD4+ T cells on the immune system, clinical experience with tumor-specific CD4+ Th cells is almost non-existent. Unlike most other tissues, bone marrow-derived cells constitutively express MHC class II and CD4+ T cells play crucial role in mediating the curative GVL effect after allogeneic SCT and donor lymphocyte infusion (DLI). Furthermore, experimental evidences suggest that MHC class II-restricted antigenic targets recognized by CD4+ T cells exist in both solid cancers and in hematological malignancies. Therefore adoptive immunotherapy using CD4+ T cells in the setting of leukemia might be especially relevant. The goal of this study is to establish a simplified non-individualized protocol of generating LAA-reactive CD4+ T cells from patients and normal donors for adoptive immunotherapy directed against common leukemia-associated antigens (LAA) expressed in acute myeloid leukemias (AML) and myelodysplastic syndrome (MDS). We isolated naïve and memory CD4+ T cells from 3 normal donors and stimulated with twice at weekly interval with autologous monocytes pulsed with libraries of overlapping 15-amino acid length peptides (pepmixes) derived from WT-1, MAGE A3 and A4, PRAME and SSX2 antigens. At the end of the experiment CD4+ T cells were evaluated for reactivity against each LAA by analyzing their ability to specifically release cytokines (IL-2, TNF-α, and IFNγ) using flow cytometry. LAA-specific cells were found in either naïve or memory-derived CD4+ T cells upon stimulation with relevant pepmixes in all donors tested. However specific cytokine production could not be demonstrated when the same T cells were exposed to LAA-transduced autologous targets (LCL and T cells), raising the possibility that the majority of pepmix-reactive cells recognized epitopes that were not naturally processed. Therefore, as an alternative strategy to induce LAA-specific cells capable of targeting only therapeutically-relevant epitopes, we used autologous dendritic cells (DCs) transduced with a lentiviral vector encoding MAGE A3 antigen. Autologous CD4+ T cells were stimulated with MAGE A3 or mock-transduced DCs at an interval of 7-10 days and tested for their antigen-specific cytokine secretion. At the end of the culture we observed that Th cells expanded in presence of MAGE A3-expressing DCs and contained a significant number of cells possessing specific reactive against MAGE A3 pepmix (Figure), but not to unrelated antigenic targets, suggesting induction of LAA-reactivity against naturally-processed MAGE A3 epitopes. In summary, we demonstrate the feasibility of generating specific anti-tumor CD4+ T cells using autologous DCs engineered to express a full-length tumor antigen. This approach allows for selective expansion of polyclonal Th cells recognizing only naturally processed MHC class II-restricted epitopes. Therefore, this strategy circumvents the limitation inherent to usage of overlapping peptide libraries that might induce the expansion of high-avidity T cells specific to epitopes that are irrelevant to in vivo recognition of tumor targets. Furthermore, this approach does not rely on a particular pre-defined MHC class II restriction element, thus it is applicable to majority of donors or patients irrespective of their MHC haplotype. Disclosures: No relevant conflicts of interest to declare.

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T helper type 1/T helper type 2 cytokines and T cell death: preventive effect of interleukin 12 on activation-induced and CD95 (FAS/APO-1)-mediated apoptosis of CD4+ T cells from human immunodeficiency virus-infected persons.

Journal of Experimental Medicine ◽

10.1084/jem.182.6.1759 ◽

1995 ◽

Vol 182 (6) ◽

pp. 1759-1767 ◽

Cited By ~ 201

Author(s):

J Estaquier ◽

T Idziorek ◽

W Zou ◽

D Emilie ◽

C M Farber ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Th2 Cytokines ◽

T Helper ◽

Th1 Cell ◽

Immunodeficiency Virus ◽

Induced Apoptosis

Human immunodeficiency virus (HIV) infection leads to a progressive loss of CD4+ T helper (Th) type 1 cell-mediated immunity that is associated with defective in vitro CD4+ T cell proliferation and abnormal T cell death by apoptosis in response to T cell receptor (TCR) stimulation. Quantification of interleukin (IL)-2, interferon gamma, IL-4, IL-5, and IL-10 secretion by immunoassays, and of interferon gamma, IL-4 and IL-10 messenger RNA expression by competitive reverse transcriptase polymerase chain reaction after in vitro stimulation of the TCR revealed a similar Th1 cytokine profile in T cells from HIV-infected persons and from controls. These data indicated that the loss of CD4+ Th1 cell function in HIV-infected persons is not related to a Th1 to Th2 cytokine switch as previously proposed, but to a process of activation-induced death of CD4+ Th1 cells. Despite the absence of elevated levels of Th2 cytokines, apoptosis of CD4+ T cells, but not of CD8+ T cells, was prevented in vitro by antibodies to IL-10 or IL-4, two Th2 cytokines that downregulate Th1 cell responses, or by the addition of recombinant IL-12, a cytokine that upregulates Th1 functions. TCR-induced apoptosis of T cell hybridomas and preactivated T cells has been shown to involve the CD95 (Fas/Apo-1) molecule. CD4+ and CD8+ T cells from HIV-infected persons expressed high levels of the CD95 molecule, and, in contrast to T cells from controls, were highly sensitive to antibody-mediated CD95 ligation, which induced apoptosis in a percentage of T cells similar to that induced by TCR stimulation. As TCR-induced apoptosis, CD95-mediated apoptosis of CD4+ T cells, but not of CD8+ T cells, was prevented by the addition of recombinant IL-12. Together, these findings suggest that apoptosis of CD4+ T cells from HIV-infected persons involves an abnormal sensitivity to CD95 ligation, and to TCR stimulation in the presence of normal levels of Th2 cytokines. The preventive effect of IL-12 on both mechanisms has potential implications for the design of immunotherapy strategies aimed at the upregulation of CD4+ Th1 cell functions in AIDS.

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Distinctive CD26 Expression on CD4 T-Cell Subsets

Biomolecules ◽

10.3390/biom11101446 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1446

Author(s):

Oscar J. Cordero ◽

Carlos Rafael-Vidal ◽

Rubén Varela-Calviño ◽

Cristina Calviño-Sampedro ◽

Beatriz Malvar-Fernández ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Surface ◽

Cd4 T Cells ◽

Ex Vivo ◽

T Cell Subsets ◽

Effector Memory ◽

T Helper ◽

Cd26 Expression

Immune system CD4 T-cells with high cell-surface CD26 expression show anti-tumoral properties. When engineered with a chimeric antigen receptor (CAR), they incite strong responses against solid cancers. This subset was originally associated to human CD4 T helper cells bearing the CD45R0 effector/memory phenotype and later to Th17 cells. CD26 is also found in soluble form (sCD26) in several biological fluids, and its serum levels correlate with specific T cell subsets. However, the relationship between glycoprotein sCD26 and its dipeptidyl peptidase 4 (DPP4) enzymatic activity, and cell-surface CD26 expression is not well understood. We have studied ex vivo cell-surface CD26 and in vitro surface and intracellular CD26 expression and secretome’s sCD26 in cultured CD4 T cells under different polarization conditions. We show that most human CD26negative CD4 T cells in circulating lymphocytes are central memory (TCM) cells while CD26high expression is present in effector Th1, Th2, Th17, and TEM (effector memory) cells. However, there are significant percentages of Th1, Th2, Th17, and Th22 CD26 negative cells. This information may help to refine the research on CAR-Ts. The cell surface CD45R0 and CD26 levels in the different T helper subsets after in vitro polarization resemble those found ex vivo. In the secretomes of these cultures there was a significant amount of sCD26. However, in all polarizations, including Th1, the levels of sCD26 were lower (although not significantly) compared to the Th0 condition (activation without polarization). These differences could have an impact on the various physiological functions proposed for sCD26/DPP4.

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The CD28 ligand B7/BB1 provides costimulatory signal for alloactivation of CD4+ T cells.

Journal of Experimental Medicine ◽

10.1084/jem.173.3.759 ◽

1991 ◽

Vol 173 (3) ◽

pp. 759-762 ◽

Cited By ~ 201

Author(s):

L Koulova ◽

E A Clark ◽

G Shu ◽

B Dupont

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

T Lymphocyte ◽

Cd4 T Cells ◽

B Lymphocyte ◽

Interleukin 2 ◽

Class Ii ◽

Cell Surface Molecule

Activation via the T lymphocyte cell surface molecule CD28 provides a potent amplification signal for interleukin 2 (IL-2) production in several in vitro systems. The B lymphocyte activation antigen, B7/BB1, is a natural ligand for CD28. Here we investigate the role of CD28 and B7/BB1 in primary activation of CD4+ T lymphocytes stimulated with allogeneic B lymphoblastoid cell lines. A subset of peripheral CD4+ T cells that is unresponsive to crosslinking of CD3/T cell receptor (TCR) with CD3 monoclonal antibody (mAb) does proliferate in response to allogeneic B lymphoblasts. TCR binding to allogeneic major histocompatibility complex antigens was an absolute requirement for activation of these cells because mAbs to either CD3 or human histocompatibility leukocyte antigen (HLA) class II completely inhibited activation. CD28 and B7/BB1 antibodies inhibited T cell proliferation 90% and 84%, respectively. Similar results were obtained with the total CD4+ T lymphocyte population. Crosslinking of HLA-DR antigens on small, resting B cells induced rapid expression of B7/BB1, which peaked at 6 h and returned to baseline levels within 18 h. These data demonstrate that CD28-B7/BB1 binding provides an important early second signal for alloactivation of CD4+ T lymphocyte by B lymphoblasts. The results also suggest that T cells interacting with allogeneic resting B cells may induce B7/BB1 expression in the alloantigen-presenting cell as a consequence of interaction between the TCR and class II molecules.

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Expansion of immunoglobulin autoreactive T-helper cells in multiple myeloma

Blood ◽

10.1182/blood-2006-11-056242 ◽

2008 ◽

Vol 111 (5) ◽

pp. 2725-2732 ◽

Cited By ~ 14

Author(s):

Masih Ostad ◽

Margareta Andersson ◽

Astrid Gruber ◽

Anne Sundblad

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Single Cell Analysis ◽

T Helper ◽

Cell Reactivity ◽

Th Cells

Activation and expansion of T helper (Th) cells followed by regulation of activation are essential to the generation of immune responses while limiting concomitant autoreactivity. In order to characterize T cells reactive towards myeloma-derived monoclonal immunoglobulin (mIg), an autologous coculture assay for single-cell analysis of mIg-responding cells was developed. When cultured with dendritic cells loaded with mIg, CD4+ Th cells from patients with progressing multiple myeloma (MM) showed a proliferative MHC class II–dependent response. CD8+ T-cell reactivity and Th1 activation were consistently low or absent, and Th2 and regulatory cytokines were expressed. The presence of such non-Th1 CD4+ T cells in peripheral blood was independent of treatment status, while the frequencies of responding cells varied between patients and reached the same order of magnitude as those measured for tetanus toxoid–specific Th memory cells. Furthermore, investigations of T-cell subpopulations indicated a possible regulatory role on the mIg responsiveness mediated by suppressive CD25highFOXP3+CD4+ T cells. It is proposed from the present results that a predominant in vivo activation of non-Th1 mIg-reactive CD4+ T cells constitute an Ig-dependent autoregulatory mechanism in human MM, with possible tumor growth supporting or permissive effects.

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Treg-Resistant Cytotoxic CD4+ T Cells Dictate T Helper Cells in Their Vicinity: TH17 Skewing and Modulation of Proliferation

International Journal of Molecular Sciences ◽

10.3390/ijms22115660 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5660

Author(s):

Cindy Hoeks ◽

Marjan Vanheusden ◽

Liesbet M. Peeters ◽

Piet Stinissen ◽

Bieke Broux ◽

...

Keyword(s):

T Cells ◽

Conditioned Medium ◽

T Helper Cells ◽

Cd4 T Cells ◽

Cell Contact ◽

T Helper ◽

Th Cells ◽

Inflammatory Microenvironment ◽

Helper Cells

Cytotoxic CD4+ T cells (CD4 CTL) are terminally differentiated T helper cells that contribute to autoimmune diseases, such as multiple sclerosis. We developed a novel triple co-culture transwell assay to study mutual interactions between CD4 CTL, conventional TH cells, and regulatory T cells (Tregs) simultaneously. We show that, while CD4 CTL are resistant to suppression by Tregs in vitro, the conditioned medium of CD4 CTL accentuates the suppressive phenotype of Tregs by upregulating IL-10, Granzyme B, CTLA-4, and PD-1. We demonstrate that CD4 CTL conditioned medium skews memory TH cells to a TH17 phenotype, suggesting that the CD4 CTL induce bystander polarization. In our triple co-culture assay, the CD4 CTL secretome promotes the proliferation of TH cells, even in the presence of Tregs. However, when cell−cell contact is established between CD4 CTL and TH cells, the proliferation of TH cells is no longer increased and Treg-mediated suppression is restored. Taken together, our results suggest that when TH cells acquire cytotoxic properties, these Treg-resistant CD4 CTL affect the proliferation and phenotype of conventional TH cells in their vicinity. By creating such a pro-inflammatory microenvironment, CD4 CTL may favor their own persistence and expansion, and that of other potentially pathogenic TH cells, thereby contributing to pathogenic responses in autoimmune disorders.

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THU0032 MODIFIED PEPTIDES AS A NOVEL IMMUNOTHERAPY FOR RHEUMATOID ARTHRITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.2714 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 229.1-229

Author(s):

E. Araklioti ◽

L. Herman ◽

N. Q. N. Nguyen ◽

R. Roohi Ahangarani ◽

M. Erak ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Flow Cytometric Analysis ◽

Class Ii ◽

Research Support ◽

Modified Peptides ◽

Apoptotic Activity ◽

Walloon Region

Background:Rheumatoid arthritis (RA) is a highly prevalent and severe systemic autoimmune disease associated with permanent disability and strong socio-economic burdens. Currently, there is no therapeutic treatment and RA patients rely on lifelong, costly treatments. Imcyse develops modified peptides eliciting antigen specific cytolytic CD4 T cells (cCD4+) that induce apoptosis of antigen presenting cells (APC) in a contact dependent manner. cCD4+ also induce apoptosis of autoantigen-specific bystander T-cells, activated by the same APC thus eliminating the risk of general immunosuppression. Peptides consist of MHC class II T cell epitopes of a target autoantigen modified in their flanking region by the addition of an amino acid sequence containing a thiol-disulphide oxidoreductase active motif1.Objectives:The goal of this study was to synthesize modified peptides from a target RA autoantigen and test their potency to generatein vitrospecific and cytolytic CD4+ T cells from RA patients.Methods:We designed modified peptides from a target RA autoantigen after in silico and in vitro assessment to identify MHC II core binding region, HLA class II binding capacities and physiochemical properties.CD4+ T cells were purified from PBMC of a newly diagnosed seropositive RA patient and co-cultured with autologous APC in the presence of the modified peptide. The CD4+ T cells were restimulated periodically. Peptide’s ability to generate specific CD4+ T cells was evaluated by flow cytometric analysis of the expression of surface activation marker CD154 (CD40L). The peptide specific CD4+ T cell lines were sorted based on their surface CD154 expression. Their pro-apoptotic activity was assessed after overnight (O/N) co-culture of CD4+ T cells with fluorescent tracer labelled autologous lymphoblastoid cells lines (LCL). Flow cytometry quantification of LCL apoptosis was measured by annexin V staining. MHC II restriction of CD4+ T cells was demonstrated by the addition of blocking antibodies against HLA-DR, DP or DQ molecules.Results:CD4+ T cells were in vitro expanded after six consecutive stimulations with the peptide. We investigated their specificity by flow cytometry and showed that 69% of CD4+ T cells that were stimulated O/N in the presence of the peptide expressed the activation marker CD154 versus 29% of CD4+ T cells that were stimulated in its absence. These cells were sorted based on CD154 expression following specific stimulation. Cell enrichment was then assessed by flow cytometric analysis. Data showed that more than 91% (background 3%) were peptide specific based on CD154 expression.After co-culture of CD4+ T cells with LCL, in independent experiments, Annexin V binding was detected on peptide loaded LCL, ranging from 69% to 89%, while when LCL were kept unloaded these values were between 30% and 55%, respectively, indicating that when specifically activated, these CD4+ T cells had pro-apoptotic activity. When both the peptide and blocking antibodies against HLA-DR, DP or DQ molecules added in the co-culture the pro-apoptotic activity was inhibited by 68%, 20% and 25%, respectively.Conclusion:The preliminary but very promising data show that our modified peptide generates peptide-specific CD4+ T cells with lytic properties that lyse target APC in an HLA class II specific manner. Our plan is to show that these CD4+ T cells can also induce apoptosis in bystander T cells and to further validate our results in additional RA donors.References:[1]Carlier, V. A., Vanderelst, L., Janssens, W. & Jacquemin, M. G. Increased Synapse Formation Obtained by T Cell Epitopes Containing a CxxC Motif in Flanking Residues Convert CD4 + T Cells into Cytolytic Effectors.7, (2012).Disclosure of Interests:Eleni Araklioti Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Ludivine Herman Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Ngoc Quynh Nhu Nguyen Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Roxana Roohi Ahangarani Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Milos Erak Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Bernard Lauwerys: None declared, Patrick Durez Speakers bureau: AbbVie, Bristol-Myers Squibb, Celltrion, Eli Lilly, Pfizer, Sanofi, Vincent Geenen: None declared, Aaron Winkler Shareholder of: Shareholder of Pfizer, Inc, Employee of: Full time employee of Pfizer, Inc, Marcelle Van Mechelen Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Luc Vander Elst Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Vincent Carlier Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region

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CD4+ T-cell epitopes associated with antibody responses after intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice

Blood ◽

10.1182/blood-2011-08-374645 ◽

2012 ◽

Vol 119 (17) ◽

pp. 4073-4082 ◽

Cited By ~ 39

Author(s):

Katharina N. Steinitz ◽

Pauline M. van Helden ◽

Brigitte Binder ◽

David C. Wraith ◽

Sabine Unterthurner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Class Ii ◽

Structural Features ◽

Cd4 T Cell ◽

T Cell Epitopes ◽

Mhc Class Ii Molecules

Abstract Today it is generally accepted that B cells require cognate interactions with CD4+ T cells to develop high-affinity antibodies against proteins. CD4+ T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4+ T cells that can bind to the MHC class II–peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4+ T-cell epitopes presented by HLA-DRB1*1501 to CD4+ T cells during immune responses against FVIII. CD4+ T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.

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