T cell cloning, cloning of alloreactive human t cells, Antigen recognition by B and T cell receptor, T cell subtypes

cloning conditions, media used, cloning conditions, characterization of T cell clones, role of mhc 2 and antigens in T cell cloning, applications of T cell cloning in vaccine development.

1. BANGALORE UNIVERSITY Dept. of microbiology and Biotechnology SEMINAR PRESENTATION TOPIC: T cell cloning Guided by, Dr.S . T. GIRISHA SIR Associate professor Dept. of Biotechnology J B Campus P resented by , VARUN H R 19TUST4040 2 nd semester M.sc biotechnology JB campus

2. CONTENTS T-Cell cloning. General cloning conditions. Media used for cloning and culture. Cloning of Human alloreactive T cells. Cloning of Human T cells reactive with soluble antigens. Antigen recognition by T cell and B cell receptors. Role of Antigens and the MHC class II in T cell cloning. Immunologically relevant antigens. T cell subtypes. Applications of T cell cloning in vaccine development.

3. T Cell cloning The approach for or obtaining large number of identical functional cells is the derivation of clones of normal T cells. It is now possible to obtain easily cloned T cells which can be maintained in culture indefinitely CLONES: Clone will consist of cells , which share the same phenotypic characteristics and functions. T cell cloning was 1 st discovered by Gillis et.al. in 1978.

4. GENERAL CLONING CONDITIONS Two other distinct approaches have been used to derive and maintain Human and Murine T cell clones. The 1 st employs IL-2 alone as the stimulus for T cell growth following t cell activation. Corresponding lymphoid cells, sometimes collected after initial stimulation with antigen in-vivo are placed in culture with the appropriate antigen. The cultured cells are then fed only with IL-2; with this approach it is usually necessary to passage is in weak cultures for several weeks before it is possible to isolate clones with a reasonable success rate.

5. Since the frequency of T cells capable of growing in IL-2 alone seems to be low, properties of cloned T cells derived and maintained by culture in IL-2 alone maybe unstable. Such cloned cells also may have lost responsiveness to specific antigenic challenge . 2. T he 2 nd approach for deriving and maintaining T cell clones employs stimulating antigen and filler cells in addition to IL-2. Alloreactive clones can be derived from primary MLR using this approach, and cloning efficiency from secondary culture may approach 100%.

6. In this case of cloned T cells reactive with “conventional” soluble antigens, the filler cells provide a source of Ia - positive antigen presenting cells which are required for T cell stimulation. These two different approaches for deriving and maintaining cloned T lymphocytes yield cells that may have rather different properties. The method of choice will depend on its proposed use of the cell clones.

7. MEDIA USED FOR CLONING AND CULTURE Several different culture media have been used by different investigators with generally comparable results. 1.Dulbecco’s modified eagle medium (DMEM): sometimes in addition or added nutrients has been used often for culturing Murine lymphoid cells , high glucose formulation (4500mg/ litre ) should be used. This medium is isotonic with rat and mouse cells. DMEM

8. 2. RPMI-1640 MEDIUM: Roswell park memorial medium. It has been employed for both mouse and human cell cultures. It is isotonic in human serum but it is hypotonic with respect to mouse and rat serum, if used for culture of rodent cells. It may be preferable to prepare cell suspensions using Hanks balanced salt solution or other compound medium isolate in mouse serum to avoid lysis and formation of a DNA gel which may trap cells. RPMI

9. TYPES OF T CELL CLONING THERE ARE MAINLY TWO TYPES OF T CELL CLONING Alloreactive T cell cloning. Antigen specific T cell cloning.

10. CLONING OF HUMAN ALLOREACTIVE T CELLS Isolate “ peripheral mononuclear blood lymphocytes” (PMBL) by ‘ Ficoll-Hypaque density gradient centrifugation’. Cells to be cloned are activated in primary MLR (mixed lymphocyte reaction) using 5x10^-1 responding and 5x10^6 irradiated stimulating cells in 10ml of MLR medium. Complete medium contains RPMI-1640 supplemented with 10% FCs (calf serum) or human male AB serum, antibiotics (penicillin / streptomycin), 0.2 mM glutamin and 20 mM HEPES buffer.

11. Cells are cultured in 10ml of medium or by doubling the cell numbers in 20ml of medium. Cells can be cultured upright in a 70ml- falcon flask for 2 weeks at 37ºc in a humidified atmosphere containing 7.5% co2. Recover surviving primed responder cells by density gradient centrifugation over Ficoll - hypaque and restimulated with fresh irradiated stimulator cells in fresh culture medium at a responder / stimulator ratio of 1:5. By repeating stimulations a polyclonal line for alloantigens present on the stimulator cells and not present on the responder cells can be established.

12. At the time of cloning, 4days following serial restimulation , T cells are isolated as blasts by centrifugation over discontinuous percoll gradients and cloned by limit dilution . Gradients are generally between 70 and 34%, percoll gradient centrifuged at 450G for 10mins and T cell blasts are recovered in the interface between the 40&50% percoll layers. Isolated T cell blasts are washed once in culture medium and plated at 10^3cells/well to give a sufficiently low probability of non clonal growth in any one well.

13. The cloning medium consists of complete medium but supplemented with IL-2 as a concentration found to be optimal to support growth of IL-2 dependent line. T cells are seated in flat bottomed 96 well plate ( microtiter ) together with 3x10^5 cells/well of irrradiated peripheral stimulator lymphocytes (PSL) in a total volume of 200 µl of MLR medium. These microcultures are fed each 5 days by adding 50 µl of fresh cloning medium. Cell growth is determined by visual inspection using an inverted phase microscope. After 1-2 weeks, the cultures are transferred to 24 well culture plates with fresh stimulater cells ( 1x10^-6 PMBL/ml of cloning medium).

14. Ficoll hyapaque density gradient centrifugation percoll gradient centrifugation Microtiter falcon flask Tcell blasts

15. FUCTIONAL CHARACTERIZATION OF T CELL CLONES T Cell clones will be either cytolytic or will proliferative and not be cytolytic . Generally cytolytic clones are specific for MHC class 1 antigens, whereas most alloproliferative noncytolytic clones are directed against MHC class 1 antigens. Alloreactivity is coexisted by incorporation of tritiated thymidine in a proliferation assay. For cytolytic activity T cell clones are sheered in standard 4-hours chromium-51 release assay .

16. T cell clones either cytolytic or proliferating noncytolytic can be freezed using standardised freezing techniques. Briefly, in hour after antigen exposure clones are harvested and resuspended in pre cooled RPMI supplemented with 20% FCs and 10% DMEM . Cells are frozen at a rate of approximately 1ºc , minuted by placing them in a styrofoam box in a -70ºc. For freezer cells, however should be served in liquid nitrogen. Styrofoam box

17. PMBL( isolated from ficolll hypaque centrifugation) Activated in MLR Responding + stimulating cells [in 10ml MLR medium] Doubling number 20ml of MLR medium Cultured upright 70ml- falcon flask [ for 2 weeks at 37c + 7.5% co2] centrifugation Responder cell + fresh irradiated stimulator cells : fresh culture medium (1:5) restimulatins Polyclonal line Alloantigens on percoll stimulator cells gradients T cell blasts limit dilution Wells containing cloning medium+ IL-2 T cells are setteled 96 well plate( microtitre ) + irradiated PSL [ 200 µl ] 50 µl medium, 5D Visual inspection [ inverted phase microscope ] Proliferation / cytolytic test freezed A lloreactive T cell cloning

18. CLONING OF HUMAN T CELLS REACTIVE WITH SOLUBLE ANTIGEN Basically the technology for cloning antigen specific human T cells does not differ appreciably from that described for cloning human alloreactive T cells. The only difference is that addition of exogenous antigen at each restimulation and the need for haplotype antigenic presenting cells to drive T cell proliferation. Antigen concentration in the range of 10 µM is generally sufficient to induce proliferation at each step of the T cell expansion and cloning.

19. ANTIGEN RECOGNITION BY B CELL AND T CELL RECEPTORS

20. ANTIGEN RECOGNITION BY T CELLS In contrast to the Ig which interact with pathogens & their toxic product in extracellular spaces of the body. T cells recognise foreign Ags that are displayed on the surfaces of the body’s own cells. T cells can detect the presence of an intracellular pathogen because infected cells display Peptide fragments derived from the pathogen’s proteins on their surface .. These foreign peptides are delivered to the cell surface by specialised host cell glycoproteins.

21. These are encoded in a large cluster of genes called MHC & peptide-binding glycoproteins The recognition of Ag as a small peptide fragment bound to an MHC molecule & displayed at the cell surface is one of the most distinctive feature of T cells.

22. TCR RECOGNIZES Ag IN THE FORM OF A FOREIGN PEPTIDE BOUND TO AN MHC MOLECULES Ag recognition by TCR clearly differs from recognition by BCR B cell recognizes Ags directly by binding of Ig to the intact Ag. Abs typically bind to the surface of protein Ags , contacting a.a that are discontinues in the primary structure but are brought together in the folded protein. T cells in contrast were found to respond to short adjacent a.a sequences in proteins. These sequences were often buried within the native structure of the protein & thus could not be recognized directly by the TCR. Unless some unfolding of the protein Ag & its ‘processing’ into peptide fragments had occurred.

23. The nature of the Ag recognized by T cells became clear with the realization that the peptides that stimulate T cells. And are recognized only when bound to an MHC molecule . The ligand recognized by the T cell is thus a complex of peptide & MHC molecule. The TCR interacts with this ligand by making contacts with both the MHC molecule & the Ag peptide.

24. ANTIGEN RECOGNITION BY B CELL The antigen recognition molecules of B cells are the Ig . These proteins are produced by B cells in a vast range of antigen specificities . Membrane bound Ig on the B cell serves as the cell’s receptor for antigen and is known as BCR The BCR controls the activation B cell. B cells are able to grab and gather Ags by engaging biochemical modules, receptor clustering, cell spreading, receptor transport & Ag presentation.

25. It is noteworthy that BCR for an Ag is significant sensor that is required for B cell activation, survival & development A B cell is activated by its first encounter with an Ag that binds to it receptor, the cell proliferates & differentiate to generate plasma B cells & memory B cells. The BCR has a crucial functions upon interaction with Ag. One function is signal transduction, involving changes in receptor oligomerizaton . The second function is to mediate processing of the Ag & presentation of peptides to helper T cells.

26. The BCR is required for normal antibody production & defect in BCR signal transduction may lead to immunodeficiency &B cell malignancy. Ig of the same antigen specificity is secreted as Ab by terminally differentiated B cell- the plasma cells The secretion of Abs, which bind to pathogens or their toxic products in the extracellular space of the body is the main effector function of B cells in adaptive immunity.

27. ROLE OF ANTIGENS AND THE MHC CLASS II IN T CELL CLONING Our understanding of the way in which the class I and class II surface glycoproteins encoded by the major histocompatibility complex (MHC) control T cell function has changed dramatically in the last 5 years. Of the greater impact has been the conversion of a number of other series of work in past five years which has convinced most researchers in the field that degraded peptide form of foreign antigens are recognized in association with the MHC products by both class I and class II restricted T cells.

28. Progress has been so rapid that today there are many examples of peptides 8-20 aminoacids in length, which provide the foreign antigen epitope for T cell clones immunised against native protein antigens. In most cases only the peptide fragment and not the native or intact protein antigen can be recognised in association with MHC. In the case of a healthy individual T helper cells are scanning class II leaving cells for novel peptide – class II complexes while cytotoxic T lymphocytes (CTLS) are scouting for novel peptide – class I complexes.

29. MHC molecules also critically involved in shaping the T cell receptor repertoire . During T cell ontogeny in the thymus; the MHC molecules of the thymic epithelial cells are involved in selecting which T cell receptors are exported to the fine periphery; epithelial class II glycoprotein select the class II restricted repertoire and epithelial class I antigens select the cytotoxic T cell repertoire. Since the discovery of MHC restriction there have been two broad classes of explanation for MHC- linked control of T cell recognition:

30. T cells recognize epitopes on the foreign antigen and on the self MHC encoded marker independently. According to this view, the T cell may have 2 receptors one for foreign epitope and one for self MHC, which can be manipulated separately. T cells have one receptor that will bind the complex of foreign antigen plus self MHC. The receptor may bind only the foreign antigen, which is held on presented by the MHC molecules in an MHC-allele specifically – “ the determining selection hypothesis”. Alternatively the receptor may bind self MHC “ modified” by the presence of the foreign antigen – ‘the altered-self hypothesis’.

31. IMMUNOLOGICALLY RELEVANT ANTIGENS Immunologically relevant antigens are the antigens which are having similar epitopes or similar structural conformations. Epitopes are the immunologically active antigen regions on a complete antigen that region actually bind to B or T cell receptor.

32. The B cell recognizes an enormous variety of epitopes these displayed on the surface of bacteria or viral particles as well as those displayed on soluble proteins, glycoproteins, polysaccharides or lipopolysaccharides that have been released from invading pathogens The T cell recognizes protein epitopes displayed together with MHC molecules on cells including altered self cells such as virus infected self cells and cancerous cells.

33. T CELL SUBTYPES There are five major types in T cell are as follows Helper T cells Cytotoxic T cells Suppressor T cells Memory cells Natural killer T cells Helper Tcells (TH) T cell displaying CD4+ generally functions as T helper cells. T helper cells recognises and interacts with an antigen and MHC class II molecular complex. There are two subtypes TH1 and TH2

34. Functions of T helper cells Activates B cells and T cytotoxic cells. Enhances macrophages. Enhance the secretion of cytokines. B. Cytotoxic T cells( Tc ) T cells displaying CD8+ generally function as T cytotoxic cells. Tc cells recognise and interacts with an antigen and MHC class I molecule complex. Finally the T cytotoxic requires exposure to IL-2 to become TH cells. Functions of cytotoxic T cell Which recognises the specific Ag molecule Stimulates immune response Helps in elimination of pathogens

35. C. Suppressor T cells( Ts ) Consist CD25+ on their surface and these are earlier known as T regulatory cells . These are subpopulations of T cells which downregulates or negative regulates the immune system. Functions of T suppressor cells Mainly helps in hypersensitivity D. Memory T cells Subset of Ag specific T cells that persist long time after infection. Memory cells may be either CD4+ or CD8+. Functions of Memory T cells Memory against past infections They quickly expand to large numbers.

36. E. Natural killer T cells ( NKT) These are not similar to the Nk cells of innate immunity. NKT cells recognize glycolipid antigen presented by a molecule called CD1d. Functions of NKT cells Perform functions ascribed to both TH and Tc cells. Recognize and eliminate tumor cells and cells infected with viruses.

37. APPLICATION OF T CELL CLONING IN VACCINE DEVELOPEMENT Through the use of antigen specific T cell clone , and the extended culture of B l ymphocytes , major advances have been made in understanding the complex cellular and humoral interactions that govern an immune response. T cell clones have helped in defining the molecular basis for the phenomenon of MHC restricted recognition of foreign antigen. T cells recognise the protein antigen only in the presence of APCs.

38. Evidence that CTLS could recognize viral antigens that were not integral membrane proteins was came first from the study of cloned T cells isolated following immunization with ‘ influenza virus’. T cell clones have also proved useful for characterizing the role of particular cell surface molecule in cell function. The frequency of T cells that recognize and respond to MHC antigens of other members of the species is extraordinarily high. T cell clones finds application in the development of vaccine against Diabetes, HIV virus.

39. T cell clones are developed for particular Ag by which the diseases are caused by that Ag are avoided by this technique. T cell clones helps to control the Hypersensitivity and allergic reactions. T cell clones are used in immunotherapy of intracellular bacterial infection. Current approaches are made in the development of vaccines against Enteric disease and also for Tuberculosis and cancer

40. CONCLUSION Certain cloning conditions has to be maintained for efficient cloning like IL-2, stimulating antigen and filler cells. Appropriate media should be used for good cloning like, DMEM, RPMI 1640. Procedure includes use many centrifugation techniques like ficoll-hyapaque density gradient and percoll gradient centrifugation. It also includes antibiotics, buffer, limit dilution. Cytolytic clones are specific for MHC class I Ag, proliferative noncytolytic clones are directed against MHC class I Ag. B and T cells will recognize the Ags with the help of receptor on their cell surface and produces specific Abs to destroy them.

41. B cell recognises Ag and destroys them by secreting Ab T cell recognizes short amino acid sequences burried inside protein and they have to be exposed outside for T cells to act on them. TH cells scans for class II, TC cells for class I complexes . MHC helps in shaping T cell receptor repertoire. Ags with similar epitopes are called immunologically relevant Ags and this epitope region binds to B or T cell receptor. T cell clones finds many advantages in vaccine development, hypersensitivity, and immunotherapy.

42. REFERENCES Sabrina mariotti , Generation of human T cell clones, Article in methods in molecular biology- February 2009. William E., M d. Paul, fundamental immunology 5 th edition, Lippincott Williams and Wilkins publishers. Janeway ca jr .,travers P, walport M et .al, Immunobiology : The immune system in health and disease, 5 th edition, new york ; garland science : 2001.

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