The Impact of Interleukin 2 on Rapid T Cell Expansion

The impact of Interleukin 2 on rapid T cell expansion

Arian Sadeghi

Project Report 20p MN3 Biology / molecular biology

Department of clinical immunology

Uppsala University Hospital

“Science is nothing but developed perception, interpreted intent, common sense rounded out and minutely articulated”.

George Santayana (1863-1952)

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Index:

1.0 The immune system 4

1.1 Innate and adaptive immunity 4

1.2 Adaptive immune response 4

2.0 The major histocompatibility complexes and antigen presentation 5

3.0 T lymphocyte activation 7

4.0 Immunotherapy 8

4.1 Cancer vaccines 8

4.2 Dendritic cells 9

4.3 Viral vectors 9

4.4 Adoptive cell transfer therapy 10

4.5 Adoptive T cell transfer therapy for treatment of EBVand CMV 11

4.6 Adoptive T cell transfer for treatment of melanoma 12

5.0 Material and methods 14

5.1 Isolation and expansion of CMV specific CD8+ T cells 14

5.1.1 Separation of lymphocytes and monocytes 14

5.1.2 Differentiation and maturation of dendritic cells 14

5.1.3 Generation of CMV-directed T cells 14

5.2 Stimulation of mononuclear cells with irradiated autologous LCLs 14

5.3 Isolation and expansion of TIL microcultures from tumor tissue 15

5.4 Rapid Expansion Protocol 15

5.5 Tetramer analysis 16

5.6 Intercellular interferon gamma staining of T cells 16

6.0 Results 17

6.1 Generation of dendritic cells from monocytes 17

6.2 Generation of Cytotoxic T lymphocytes specific for CMV pp65495-503

peptide using peptide loaded mature DC 18

6.3 Rapid expansion of CMV restricted T cells 19

6.4 Generation and expansion of EBV specific T cells 23

6.5 Rapid expansion of TILs 23

7.0 Discussion 25

8.0 Future perspectives 26

9.0 References 27

Abbreviations

ACT Adoptive cell transfer therapy

CTL Cytolytic T lymphocyte

CMV Cytomegalovirus

CpG Cytosine-phosphate-Guanine

DC Dendritic cell

EBV Epstein-Barr virus

ER Endoplasmic reticulum

FITC Flourescein-isothiocyanate

GM-CSF Granulocyte macrophage colony stimulating factor

GVHD Graft versus host disease

HLA Human leukocyte antigen

IFN Interferon

IL Interleukin

imDC Immature dendritic cell

MHC Major histocompatibility complex

PBMC Peripheral blood mononuclear cell

PE phycoerythin

PerCP Peridinin chlorophyll protein

TAP Transporters associated with antigen processing

TCR T cell receptor

TGF Tissue growth factor

TIL Tumor infiltrating lymphocyte

TNF Tumor necrosis factor

Abstract

In this work isolation and rapid expansion of cytotoxic antigen specific CD8+ T cells have been studied. The T cells used were directed against Cytomegalovirus, Epstein-Barr virus and melanoma, since such T cells have been adoptively transferred to treat patients in numerous clinical trails. In many of these clinical trails the T cells have been expanded to clinical relevant numbers using an agonistic anti-CD3 antibody, IL-2 and irradiated allogenic feeder cells before transfer. The focus has been on increasing T cell numbers with sustained phenotype and function using modified versions of this protocol. In particular the influence of IL-2 on T cell expansion rate, phenotype and function has been extensively studied. IL-2 is a great catalyst in T cell evolution, growth and proliferation. Results indicate that IL-2 aided the expansion of T cells but not during the whole two week expansion phase. The rapid growth of the T cells proved to have influence upon T cell phenotype whereas cell function was maintained. In conclusion, expansion probably changed the bias towards function as to phenotype.

1.0 The immune system

The goal of the living is to survive and to preserve life and to this end an organism must be able to distinguish between self and non-self. Non-self in this case is the actual physical surrounding of the organism e.g. dust, pollens, microorganisms, drugs, chemicals, etc. Therefore, protection against such agents is an absolute necessity for survival and an elaborated systematic defense, namely the immune system has evolved for this purpose. The immune system is built up of defensive networks and barriers spread all over the body which all collaborate in a well-orchestred manner to efficiently recognize, control and dispose of foreign matters whenever such gain accesses into the body.

1.1 Innate and adaptive

The immune system can be divided into innate and adaptive immunity. The innate immunity exists and acts without memory of previous pathogenic encounters. It is manifested in form of cellular and biochemical mechanisms that reacts rapidly to infections. Such reactions are always constant and in the same manner no matter how repetitious an infection might be. Examples of innate immunity are the skin/surface barriers including mucous membranes and cilia apparatus. The phagocytes, natural killer cells, cytokines and interferons are other examples of innate immunity. Adaptive immunity in contrast is stimulated by exposure to foreign agents and the response escalades with each successive exposure. Such increase in magnitude is due to the ability of the adaptive immunity to keep a record of previous convergences with harmful pathogens1. This delicate specificity and adapting ability is not only due to the power of remembering and acting more vigorously on second encounters, but also on expanded capacity to remember different antigens and the ability to distinguish between closely related molecules or microbes. Adaptive immunity is thus antigen-specific and the response elicited is solely depending on the type of antigen and the number of pervious encounters1. The adaptive immunity is divided into two subtypes; humoral immunity and cell-mediated immunity. Humoral immunity is based on antibody producing B lymphocytes, which recognize a specific antigen, neutralize it or tag it for destruction by other cells or mechanisms. Antibodies are abundant, of enormous variation and highly specialized. Different antibodies can elicit different responses e.g. phagocytosis or release of inflammatory mediators. The limitation of humoral immunity is that it only acts extracellulary. The cellular immunity is mediated by T lymphocytes and deals with viruses and bacteria that survive and proliferate inside host cells. T lymphocytes are divided into Helper T and Cytolytic T cells. Helper T cells are activated upon antigen recognition and in turn activate other immune cells like phagocytes and cytolytic T cells. Activated cytolytic T cells can subsequently kill target cells upon antigens recognition and as such eliminate the source of a possible infection.

1.2 Adaptive T cell response

Lymphocytes mature in generative lymphoid tissue where they are presented to the “self-antigens” in the absence of other antigens and subsequently self-reactive T cells are deleted. Maintenance of self-tolerance is a fundamental property of the immune system and failure in establishing self-tolerance leads to autoimmune diseases. After maturation the T lymphocytes leave the lymphoid organs and enter circulation. Once in circulation antigen specific clones might be activated by their specific antigens. If the initial antigen specific signal is followed by a second signal, originally generated by the innate immune system, an antigen specific immune response is initiated. This also ensures that a T cell response is triggered at the correct location i.e. the inflammatory effect. The T cell response to antigen and inflammation is cellular proliferation and differentiation into effector and memory T cells.

2.0 The major histocompatibility complexes and Antigen presentation

Cell-associated antigens must be displayed and presented for T cells recognition/activation. This task is performed by proteins encoded by genes in the major histocompatibility complex (MHC) loci. There are two main types of MHC molecules; class I and class II and they present antigens from different sources. MHC class I predominantly presents antigens originating from cytosolic proteins whereas class II presents antigens originating from extra cellular compartments (Figure 1 and Figure 2).

The MHC class I molecule in humans is known as HLA-ABC and is the product of one of the most polymorphic loci in the genome. The molecule consists of an MHC coded α-chain of ~45 kD and a non MHC coded β2-microglobulin chain. CD8+ T cells are the cells that recognize these molecules and the antigen they present. All nucleated cells, except spermatocytes, express MHC class I and can present associated peptides. All intracellular proteins become proteolytically degraded by the proteasome through ubiquitination tagging. The proteasome cleaves the protein into peptides and peptides of 6-30 residues are transported from the cytosol into the ER by the TAP (Transporters associated with antigen processing) proteins. The peptides are subsequently loaded into the peptide binding cleft of the MHC class I molecules, which are produced inside the ER. Peptide/MHC class I complex is next transported through the Golgi by exocytic vesicles to the cell surface where they interact with CD8+ T cells.

The MHC class II is known as HLA-DR/DQ in humans and consists of highly polymorphic α and β chains ~30-34 kD. These molecules exist only on professional antigen presenting cells like dendritic cells, phagocytes and B lymphocytes and are recognized by CD4+ T cells. MHC class II presents antigens originating from the extra cellular environment. Initially, professional antigen presenting cells endocytose extra cellular proteins into endosomal vesicles. These proteins are subsequently degraded into peptides by lysosomal proteases. MHC class II molecules are produced inside the ER and transported through the cytosol by exocytic vesicles. Such vesicles merge with the endosomal/lysosomal antigenic peptide containing vesicles and peptides (15-24 residues) are loaded into the peptide binding cleft of the MHC class II molecules, which are subsequently transported to the cell surface (figure 2). When a professional APC phagocytose surrounding antigens a process known as cross-presentation might occur. In this process extra cellular antigens are presented by MHC class I molecules2. Cross-presentation is only preformed by dendritic cells. Likewise, DCs are able to present endogenous antigens on MHC class II molecules3.

3.0 T lymphocyte activation

T cells use membrane proteins for antigen recognition, signal transduction and adhesion as depicted by figure 3. Different antigens are distinguished by the heterodimeric T cells receptor (TCR) consisting of the α and β chains4. Proteins responsible for signal transduction come in great variation depending on the signal being transmitted. Common for the T cells are the CD3 and ξ proteins that are non-covalently associated with the TCR and when activated by TCR antigen recognition lead to general T cell activation.

The CD4 and CD8 molecules are distinguishing factors between T cell subtypes. The CD4 is a 55-kD monomer that recognizes peptide parts of the MHC class II molecule. The CD8 molecule is a αβ or αα dimer, and recognize the MHC class I molecule5. Other accessory molecules necessary for T cell function are adhesion molecules that facilitate the migration and docking of the T cell with antigen presenting cells. Examples of adhesion molecules are: integrins and selectins6.The CD28 molecule on T cells provides the second signal needed for full T cell activation. This signal occurs when the CD28 molecule is associated with its ligand the B7-1/B7-2 (CD80 and CD86) molecules on professional APC.

The initial response from T cells upon antigen recognition is clonal expansion and differentiation into effector cells. This is facilitated by secretion of cytokines (IL-12 among others) in an autocrine fashion and through direct costimulation by professional APCs in the microenvoirment. After clonal expansion and differentiation the T cells migrate to peripheral tissue where they either become effector cells or memory cells. Effector CD4+ T cells promotes the function of CD8+ T cells by releasing immunostimulatory cytokines like IL-2. In addition, effector CD4+ T cells, activate macrophages and antibody producing B cells. Effector CD8+ T cells directly kill antigen displaying target cells in a MHC class I-antigen derived peptide-TCR specific manner.

Activated CTLs secrete cytotoxic granule proteins that trigger apoptosis in the target cells. Expression of Fas ligand is another mechanism by which the CTLs can destroy antigen displaying target cells. Binding of the Fas ligand to its target Fas protein, expressed on most cells, results in apoptosis of the target cell. A fraction of the antigen stimulated T cells develop into memory T cells, which live longer than the effector cells and do not multiply. Acceleration and refinement of a secondary immune response on subsequent infection is among the tasks of these cells.

4.0 Immunotherapy

Any attempt to mobilize or manipulate a patient’s immune system in order to cure or treat a disorder is referred to as immunotherapy. This approach is appropriate to help patients suffering from autoimmune diseases, chronic inflammations and infectious diseases. Immunotherapy generally divided in active and passive immunotherapy7. Examples of active immunotherapy are different therapeutic vaccines, such as peptides and protein-vaccines to mobilize patients own immune system de novo. Examples of passive immunotherapy are administration of monoclonal antibodies, cytokines or previously activated immune cells.

4.1 Cancer vaccines

The most frequently used approaches to stimulate the immune system to elicit an immune response against cancer are vaccines consisting of proteins or peptides administered together with an adjuvant. Adjuvants are compounds that provoke an inflammation where monocytes, neutrophils, T cells and other immune cells are recruited. Adjuvants can consist of bacterial cell components, immunostimulatory DNA i.e. cytosine/guanosine-rich motifs (CpG)8,9 or cytokines such as Interleukin 12 or granulocyte macrophage colony stimulating factor (GM-CSF)10. Dendritic cells, macrophages or other phagocytosing cells are activated by such adjuvants, capture the antigen, process and present it on their MHC molecules to which T cells and other effector cells respond. Some of the most extensive and successful peptide vaccinations on cancer patients are in melanoma and prostate cancer11,12. Results from these studies have revealed antigen specific immune responses, instances of complete or partial regression and prolonged survival13,14. Tumor cell-based vaccines can also be used15. In this case tumor cells extracted from biopsies or established cancer cell lines have been used as source of antigen16,17.Tumor cells have been irradiated and injected into patients with the hope to activate a cancer directed immune response. The cell-based vaccines have also been administered in combination with various adjuvants, like BCG 17. Additional strategies involve tumor cells transduced with vectors expressing different inflammation inducing genes18.

4.2 Dendritic cells

Dendritic cells (DCs) have many attributes that makes them suitable for human immunotherapy. Tumor cells or virus-infected cells express tumor associated antigens or pathogen specific peptides originating from these antigens, displayed in the cell surface by MHC molecules. However, most tumor cells or virus-infected cell can not initiate a primary T cell response due to the lack of co-stimulatory molecules. DCs have a distinct and highly regulated mechanism to capture and process antigens, migrate to sites of high lymphatic activity and optimally present antigenic peptides to lymphocytes. For this purpose DCs express a large array of T cell stimulation molecules such as CD40, CD54, CD80, CD86 in addition to MHC class I and II. DCs are also capable of antigen cross presentation and secretion of immunostimulatory cytokines. These attributes makes DCs very lucrative in active immunotherapy and they have been used in many clinical trails, primarily on cancer patients19. The antigen presenting and T cell activation abilities of matured DCs is far superior to that of immature DCs 20. DCs can be modified with Tumor antigens by many means21. DCs pulsed with viral or tumor antigenic peptides can trigger tumor or viral specific CD8+ T cell responses. Peptides pulsed onto DCs replace native peptides already bound to MHC22.DCs can also be incubated with protein antigens. Protein antigens are applicable independently of HLA restrictions and prior knowledge of peptide immunogenicity is not required. DCs can also be pulsed with tumor cell lysate. Lysates have the advantage of containing all relevant antigens and therefore no prior identification of tumor antigens are needed.23