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

UppsalaUniversityHospital

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

George Santayana (1863-1952)

1

Index:

1.0The immune system4

1.1Innate and adaptive immunity4

1.2 Adaptive immune response4

2.0The major histocompatibility complexes and antigen presentation5

3.0T lymphocyte activation7

4.0 Immunotherapy8

4.1Cancer vaccines8

4.2 Dendritic cells9

4.3Viral vectors9

4.4Adoptive cell transfer therapy10

4.5Adoptive T cell transfer therapy for treatment of EBVand CMV11

4.6Adoptive T cell transfer for treatment of melanoma12

5.0Material and methods14

5.1 Isolation and expansion of CMV specific CD8+ T cells14

5.1.1Separation of lymphocytes and monocytes14

5.1.2Differentiation and maturation of dendritic cells14

5.1.3Generation of CMV-directed T cells14

5.2Stimulation of mononuclear cells with irradiated autologous LCLs14

5.3Isolation and expansion of TIL microcultures from tumor tissue15

5.4Rapid Expansion Protocol15

5.5Tetramer analysis16

5.6Intercellular interferon gamma staining of T cells16

6.0Results17

6.1Generation of dendritic cells from monocytes17

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

peptide using peptide loaded mature DC 18

6.3Rapid expansion of CMV restricted T cells19

6.4Generation and expansion of EBV specific T cells23

6.5Rapid expansion of TILs23

7.0Discussion25

8.0Future perspectives 26

9.0References27

Abbreviations

ACTAdoptive cell transfer therapy

CTLCytolytic T lymphocyte

CMVCytomegalovirus

CpGCytosine-phosphate-Guanine

DCDendritic cell

EBVEpstein-Barr virus

EREndoplasmic reticulum

FITCFlourescein-isothiocyanate

GM-CSFGranulocyte macrophage colony stimulating factor

GVHDGraft versus host disease

HLA Human leukocyte antigen

IFNInterferon

ILInterleukin

imDCImmature dendritic cell

MHCMajor histocompatibility complex

PBMCPeripheral blood mononuclear cell

PEphycoerythin

PerCPPeridinin chlorophyll protein

TAPTransporters associated with antigen processing

TCRT cell receptor

TGFTissue growth factor

TILTumor infiltrating lymphocyte

TNFTumor necrosis factor

Abstract

In this work isolation and rapid expansion of cytotoxicantigen specific CD8+ T cells have been studied. The T cellsused 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 onincreasingT cell numberswith sustained phenotype and function using modified versions of this protocol.In particular theinfluence of IL-2 on T cell expansion rate, phenotype and function hasbeen 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 phenotypewhereas 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 organisme.g. dust, pollens, microorganisms, drugs, chemicals, etc. Therefore, protectionagainst such agents is an absolute necessity for survival and an elaborated systematic defense, namely the immune systemhas evolved for this purpose.The immune system is built up of defensive networks and barriers spread allover 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. Suchreactions are always constant and in the same manner no matter how repetitious an infection might be. Examples of innate immunity arethe 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 magnitudeis 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. Ifthe 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 IIpresents antigens originating from extra cellular compartments (Figure 1 and Figure 2).

The MHC class I molecule inhumans 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 loadedinto the peptide binding cleft of the MHC class I molecules,whichare 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 endosomalvesicles. 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 usemembrane proteins for antigen recognition, signal transduction and adhesion as depicted by figure3. 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 theMHC 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 throughdirect 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 appropriateto 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 haverevealed 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 cellsextracted 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 BCG17.Additional strategiesinvolve 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 cellsor virus-infected cells expresstumor associated antigens orpathogen specificpeptides originating from these antigens, displayed in the cell surface byMHC 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 withTumor 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 alreadybound to MHC22.DCs can also be incubated withprotein 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 theadvantage of containing all relevant antigens and therefore no prior identification of tumor antigens are needed.23

Viral and plasmid vectorsencoding tumor antigens have been used for in vivo and ex vivoimmunization. This method takes advantage of the unique antigen presenting ability of dendritic cells24.Genetically delivered antigens utilities the patients own antigen processing machinery and relevantpeptides are presented to the T cells. One advantage of this method is that no prior knowledge about immunogenic peptide epitopes are required. DNA and RNA vectors have been used for gene expression in DCs, with DNA being most frequently applied due to stability, manipulation capability and possibility to be producedin large quantities25. RNA transfection of DCs is currently being studied since it has proven advantageous in several aspects. The mRNA content of tumor cells can be isolated and amplified using PCR techniques before transfection into DCs26. Other advantages of RNA transfection is the benefit of expressing several tumor-derived genes within the DCs at the same time. This leads to the translation of several tumor antigens within the same DC. The short half-life of RNA and heterogeneous levels of intact protein expression achieved by RNA/DNA may also impose limitations.

The vaccination strategies mentioned have in many cases been successful in increasing the number of circulating antigen reactive lymphocytes. Unfortunately, the results have been highly inconsistent and only sporadic clinical responses have been reported.In melanoma for example, peptides used for vaccination did successfully generate tumor-reactive CTLs, but vaccination alone did only in veryfew cases result intumor regression30.Reasons for the lack of tumor rejection by immunized patients are not well characterized. Mechanisms that could limit the immune response and compromise the effects of reactive CD8+ T cells are the lack of T helper cells and the suppressive status of CD4+CD25+ regulatory T cells27. Furthermore, the CD8+ CTLs could be in insufficient amounts or be deficient in receptor avidity/signaling and other T cell functions. Production of immunosuppressive chemokines by the tumor cells or failure of the T cells to home to tumor areas could be other factors. In addition, tumor cells can acquire different escape mutations like loss of tumor antigen expression, loss or down-regulation of HLA-expression or acquire resistance to CTL lysis28.

4.4 Adoptive cell transfer therapy

Adoptive cell transfer (ACT) therapy has proven to be one of the most fruitful approaches to treat cancer and infectious diseases in both murine models and clinical trials29,30.The foundation of ACT therapy is based on the fact that tumor or virus antigen restricted T cells can be isolated, characterized and expanded ex vivo. The method is based on selection of T lymphocytes with high avidity for tumor-associated antigens or viral antigens, massiveex vivo cell expansionin the absence of regulatory T cells or other suppressor mechanisms and subsequent infusion.The presence of high affinity antigen-specific CD8+and CD4+ T cells is a prerequisite for successful ACT therapy and efficiency of the treatment have so far been directly correlated with the number of transferred tumor/virus antigen specific T cells.

4.5 Adoptive T cell transfer therapyfor treatment of EBV, CMV