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Fig. 7. CD4 T cells show different responses to peptide-MHC complexes depending on their state of differentiation. Explanation is in the text.
Concay

Fig. 7. CD4 T cells show different responses to peptide-MHC complexes depending on their state of differentiation. Explanation is in the text.
Concay

Summary of lessons from peptides isolated from I-Ak and I-Ag7
The HEL peptides extracted from I-Ak bearing B-lymphoma lines ( Fig. 5 ) separate in two major sets, depending in part on their sequence ( Table 2 ). The major peptide family has been described above, having the core segment from 52 to 60. This strong binding peptide family can occupy up to 10-20% of the I-Ak molecules and is clearly 'chemically dominant'. We should add that the interaction of a peptide acidic residue with the Arg alpha 52 described above and seen in Fig. 1 was proven by mutating the alpha chain Arg 52 to alanine, resulting in the loss of the peptide (105), or, alternatively, testing an HEL molecule where its Asp 52 was changed to Ala 52 and finding that it was very poorly selected.
A second set of selected HEL peptides bind 10-30 times more weakly to I-Ak and are selected in 60-300-fold smaller amounts on I-Ak (95, 97-99). The reason is one of the following:
1) either the peptides show a weak binding motif where there are asparagines interacting at the P4 pocket, with the P1 residues lacking an acidic residue, or

2) there are peptides with one or more unfavorable hindering residues.

The role of the negative or hindering residues cannot be underestimated. Indeed, with HEL, only 4 peptides from 21 potential ones having Asp were not selected, because of the presence of hindering amino acids (110).
Autologous peptides ( Fig. 5 ) follow these same chemical rules for binding to I-Ak as the HEL peptides. However, it is likely that many other factors contribute to abundance of selected peptides such as the amount of available protein and the major location inside the APC. It is becoming apparent that many class II MHC molecules contain weakly binding peptides that have a very fast dissociation time. Our thinking is that these peptides will not likely contribute much to T-cell reactivity but will serve to transiently give stability to the molecule and allow it *****rvive for enough time to exchange with incoming peptides (58, 59).
The analysis of I-Ag7 was motivated by the role of this protein in the spontaneous autoimmune diabetes of the NOD mouse (113). In I-Ag7 as well as in the two HLA-DQ diabetes-susceptible alleles, there is a 'non-Asp' residue at position beta 57 (114-117). All other class II MHC molecules from mouse and man have an aspartic acid that ion pairs with an arginine at alpha 67, forming the base of the P9 allelic site (74, 75, 116). In the mouse, a change of the Ser 57 back to Asp abolishes diabetes (117). Examination of peptides selected by both MHC molecules, the wild type I-Ag7 and the mutated one, we call it I-Ag7 PD, has given for the first time a clear appreciation of the importance of allelic differences in the specificity of peptide selection (100). In the study by Anish Suri with the MS group of Michael Gross, the peptides selected by I-Ag7 had a very distinctive sequence, most of them containing acidic residues at the carboxy end and frequently having runs of 2 or 3 acidic residues ( Fig. 6 ). The residues apparently cooperated in their interaction with the unpaired alpha Arg 67, as was proven by binding analysis. In contrast, changing the amino acids surrounding the P9 site resulted in a completely different family of peptides, this time lacking the predominantly acidic amino acids. It is our thinking that these binding features are likely to be found in beta cell proteins and will account for the propensity for diabetes of the NOD.
Summarizing this section, the rules for peptide selection, the importance of different segments of a peptide are now evident. The sequence motifs of core segments are major components that determine the amounts of selected peptides. There is much specificity by the MHC molecules in sampling the repertoire of its processed proteins.

From biochemistry of peptide selection to biology recent experiments
The relationship between antigen presentation and the response of the T cell can be explored critically, having a hard quantitative and chemical basis on peptide selection. I comment briefly on studies that we have recently done and that are based on our knowledge about the display of HEL peptide-MHC complexes in APC.
The CD4 T-cell response to peptide-MHC complex depends much on the natural history of the T cell. Dan Peterson and Rich DiPaolo just tested ex vivo the response of 3A9 T cells from transgenic mice to known amounts of complexes of 48-63-I-Ak displayed by APC. While double positive thymocytes responded in culture to as little as 2-3 complexes per APC (118, 119), single positive mature T cells required about 100-fold more complexes (see (120), which contains similar results in another system). In contrast, activated T cells were also very sensitive, responding to about 10-30 complexes per APC (121, 122) ( Fig. 7 ). We confirmed these results in vivo by making transgenic mice expressing HEL under a class II promoter, and directly quantitating the peptides displayed in thymic (and spleen) APC ( Fig. 4 ) (99). Negative selection took place to all HEL displayed peptides, even to those expressed at a few complexes per single APC (119). Thus, as postulated first by Lederberg (123), indeed, the lymphocyte goes through an exquisitely sensitive stage in its early development ( Fig. 7 ). This sensitive stage may be expected if negative selection is going to be operative against blood proteins and tissue components that circulate in very minimal amounts and which are displayed to a very small degree.
These results have led us to postulate a 'biochemical margin of safety', that there may be a significant differential between the amounts of peptide-MHC that delete centrally in the thymus and the amounts that activate in peripheral APC (118). A T cell directed against a self-peptide and not negatively selected in the thymus will require an encounter with relatively large amounts of the complex in APC of peripheral tissues in order to be spontaneously activated, or will require a process of activation brought about by con***ions of immunization such as when adjuvants are used (124).
One experimental model that supports the biochemical margin of safety concept is that of autoimmune diabetes in mice expressing HEL under the rat insulin promoter (125). The system of planting proteins or peptides in beta cell under the insulin promoter has been developed by many to study the role of lymphocytes and cytokines in beta cell pathology (126, 127). Our HEL mice spontaneously developed diabetes when crossed with 3A9 TCR transgenic mice. In this model, the beta cell expressed about 2 106 molecules of HEL, and there was very effective 'cross presentation' to APC in the draining lymph nodes. All indications suggest that the draining pancreatic node expresses sufficient complexes of the chemically dominant 46-63 peptide bound to I-Ak complex to exceed the 'safety margin' and activate the T cells, starting the autoimmune process.
The experiment of Rich DiPaolo (125) suggests that the large display of a chemically dominant peptide may well set the stage for development of the autoimmunity. This interpretation should be tested in experiments varying the amounts of HEL expressed in beta cells or using TCR transgenic mice to minor epitopes. Extrapolating to the NOD mouse, it could be that the spontaneous NOD diabetes follows a similar parameter, i.e. a beta cell peptide that is strongly selected by I-Ag7 molecules.
Knowing that the display of different HEL peptides differs by about 300-fold and that this differential is found with all APC ( Table 2 ), what is the clonal distribution after immunization with HEL in adjuvant? Is there truly immunodominance to the 48-63 family of peptides? We recently correlated the clonal response to HEL, using a sensitive limiting dilution assay (LDA) (119). We find little correlation between amounts displayed and the number of clones that are activated (128). Thus, under optimal con***ions, the response tends to equalize the large gradient of peptide-MHC complex. Further studies should identify the con***ions that influenced the response. Certainly, the idea of a dominant peptide governing the response is complex (129, 130) and needs to be placed in the context of recent biochemical results.
Finally, we comment on an important phenomenon that we have recently identified as a result of the chemical studies on selected HEL peptides, that of type A and B T cells (119, 131, 132) ( Table 3 , Fig. 8 ). Type A T cells are the conventional T cells induced by immunization with the protein, in our case HEL. Type B T cells, in striking contrast, are induced by immunization with the peptide that has the identical sequence as that resulting from the processing of the protein. These T cells are distinguished because they react with the peptide-pulsed APC but will not react with the APC cultured with the protein ( Table 3 ). In the case of HEL, we can identify and quantitate the major peptide and can confirm the lack of reactivity with the type B cells. The type B, as we have defined them, do not react with post-translational modifications of the peptide (These post-translational modifications can occur with some peptides and will induce specific T cells, but do not explain the A/B reactivity) (133). A very important manipulation in defining the type B reactivity is to isolate the peptide from the class II MHC, after processing of the protein HEL, and to then offer it to APC, this time as an exogenous protein. The type B in such a situation can react to it ( Table 3 )!
In studies in progress by Zheng Pu, Javier Carrero and Daved Fremont, we explain this differential reactivity as caused by a conformational difference in the peptide-MHC complex brought about by the site in the APC where the complex forms ( Fig. 2 ). The type A forms in the deep lysosomal type vesicle under the influence of the catalytic function of H-2 DM; it results in a fixed conformer. In contrast, the type B forms in the early compartment by peptide exchange without the participation of H-2 DM. MHC molecules that peptide-exchange bear weak peptides that have fast dissociation rates; these peptides have side chains that partially occupy the binding pockets and may be more flexible conformationally flexible. Thus, the type B result from more conformation of the peptide with a less constrained MHC molecule (134).
The type B T cells are abundant and have been identified against various peptides of HEL and also against autologous peptides. They represent a substantial number of T cells in the normal repertoire. Peterson and DiPaolo showed that such T cells escape negative selection in HEL transgenic mice (119). We are considering whether, under circumstances of inflammation in tissue where autologous peptides are generated, such T cells can be activated and constitute a component of autoimmunity. Teleologically speaking, the type B T cells should have a function, perhaps to favor interaction with small peptides derived from microbes or to represent a first line of defense in inflammation.

================
Fig. 8. Responses of type A and type B T cells to the peptide 48-61 of HEL. To the left is the response of a type A T cell to HEL and to the 48-61 peptide. The responses are about the same. On the right isthe response of a type B T cell, which recognizes weakly the processed peptide form HEL. Unpublished experiment of Zheng Pu.
=================
Concay

Summary of lessons from peptides isolated from I-Ak and I-Ag7
The HEL peptides extracted from I-Ak bearing B-lymphoma lines ( Fig. 5 ) separate in two major sets, depending in part on their sequence ( Table 2 ). The major peptide family has been described above, having the core segment from 52 to 60. This strong binding peptide family can occupy up to 10-20% of the I-Ak molecules and is clearly 'chemically dominant'. We should add that the interaction of a peptide acidic residue with the Arg alpha 52 described above and seen in Fig. 1 was proven by mutating the alpha chain Arg 52 to alanine, resulting in the loss of the peptide (105), or, alternatively, testing an HEL molecule where its Asp 52 was changed to Ala 52 and finding that it was very poorly selected.
A second set of selected HEL peptides bind 10-30 times more weakly to I-Ak and are selected in 60-300-fold smaller amounts on I-Ak (95, 97-99). The reason is one of the following:
1) either the peptides show a weak binding motif where there are asparagines interacting at the P4 pocket, with the P1 residues lacking an acidic residue, or

2) there are peptides with one or more unfavorable hindering residues.

The role of the negative or hindering residues cannot be underestimated. Indeed, with HEL, only 4 peptides from 21 potential ones having Asp were not selected, because of the presence of hindering amino acids (110).
Autologous peptides ( Fig. 5 ) follow these same chemical rules for binding to I-Ak as the HEL peptides. However, it is likely that many other factors contribute to abundance of selected peptides such as the amount of available protein and the major location inside the APC. It is becoming apparent that many class II MHC molecules contain weakly binding peptides that have a very fast dissociation time. Our thinking is that these peptides will not likely contribute much to T-cell reactivity but will serve to transiently give stability to the molecule and allow it *****rvive for enough time to exchange with incoming peptides (58, 59).
The analysis of I-Ag7 was motivated by the role of this protein in the spontaneous autoimmune diabetes of the NOD mouse (113). In I-Ag7 as well as in the two HLA-DQ diabetes-susceptible alleles, there is a 'non-Asp' residue at position beta 57 (114-117). All other class II MHC molecules from mouse and man have an aspartic acid that ion pairs with an arginine at alpha 67, forming the base of the P9 allelic site (74, 75, 116). In the mouse, a change of the Ser 57 back to Asp abolishes diabetes (117). Examination of peptides selected by both MHC molecules, the wild type I-Ag7 and the mutated one, we call it I-Ag7 PD, has given for the first time a clear appreciation of the importance of allelic differences in the specificity of peptide selection (100). In the study by Anish Suri with the MS group of Michael Gross, the peptides selected by I-Ag7 had a very distinctive sequence, most of them containing acidic residues at the carboxy end and frequently having runs of 2 or 3 acidic residues ( Fig. 6 ). The residues apparently cooperated in their interaction with the unpaired alpha Arg 67, as was proven by binding analysis. In contrast, changing the amino acids surrounding the P9 site resulted in a completely different family of peptides, this time lacking the predominantly acidic amino acids. It is our thinking that these binding features are likely to be found in beta cell proteins and will account for the propensity for diabetes of the NOD.
Summarizing this section, the rules for peptide selection, the importance of different segments of a peptide are now evident. The sequence motifs of core segments are major components that determine the amounts of selected peptides. There is much specificity by the MHC molecules in sampling the repertoire of its processed proteins.

From biochemistry of peptide selection to biology recent experiments
The relationship between antigen presentation and the response of the T cell can be explored critically, having a hard quantitative and chemical basis on peptide selection. I comment briefly on studies that we have recently done and that are based on our knowledge about the display of HEL peptide-MHC complexes in APC.
The CD4 T-cell response to peptide-MHC complex depends much on the natural history of the T cell. Dan Peterson and Rich DiPaolo just tested ex vivo the response of 3A9 T cells from transgenic mice to known amounts of complexes of 48-63-I-Ak displayed by APC. While double positive thymocytes responded in culture to as little as 2-3 complexes per APC (118, 119), single positive mature T cells required about 100-fold more complexes (see (120), which contains similar results in another system). In contrast, activated T cells were also very sensitive, responding to about 10-30 complexes per APC (121, 122) ( Fig. 7 ). We confirmed these results in vivo by making transgenic mice expressing HEL under a class II promoter, and directly quantitating the peptides displayed in thymic (and spleen) APC ( Fig. 4 ) (99). Negative selection took place to all HEL displayed peptides, even to those expressed at a few complexes per single APC (119). Thus, as postulated first by Lederberg (123), indeed, the lymphocyte goes through an exquisitely sensitive stage in its early development ( Fig. 7 ). This sensitive stage may be expected if negative selection is going to be operative against blood proteins and tissue components that circulate in very minimal amounts and which are displayed to a very small degree.
These results have led us to postulate a 'biochemical margin of safety', that there may be a significant differential between the amounts of peptide-MHC that delete centrally in the thymus and the amounts that activate in peripheral APC (118). A T cell directed against a self-peptide and not negatively selected in the thymus will require an encounter with relatively large amounts of the complex in APC of peripheral tissues in order to be spontaneously activated, or will require a process of activation brought about by con***ions of immunization such as when adjuvants are used (124).
One experimental model that supports the biochemical margin of safety concept is that of autoimmune diabetes in mice expressing HEL under the rat insulin promoter (125). The system of planting proteins or peptides in beta cell under the insulin promoter has been developed by many to study the role of lymphocytes and cytokines in beta cell pathology (126, 127). Our HEL mice spontaneously developed diabetes when crossed with 3A9 TCR transgenic mice. In this model, the beta cell expressed about 2 106 molecules of HEL, and there was very effective 'cross presentation' to APC in the draining lymph nodes. All indications suggest that the draining pancreatic node expresses sufficient complexes of the chemically dominant 46-63 peptide bound to I-Ak complex to exceed the 'safety margin' and activate the T cells, starting the autoimmune process.
The experiment of Rich DiPaolo (125) suggests that the large display of a chemically dominant peptide may well set the stage for development of the autoimmunity. This interpretation should be tested in experiments varying the amounts of HEL expressed in beta cells or using TCR transgenic mice to minor epitopes. Extrapolating to the NOD mouse, it could be that the spontaneous NOD diabetes follows a similar parameter, i.e. a beta cell peptide that is strongly selected by I-Ag7 molecules.
Knowing that the display of different HEL peptides differs by about 300-fold and that this differential is found with all APC ( Table 2 ), what is the clonal distribution after immunization with HEL in adjuvant? Is there truly immunodominance to the 48-63 family of peptides? We recently correlated the clonal response to HEL, using a sensitive limiting dilution assay (LDA) (119). We find little correlation between amounts displayed and the number of clones that are activated (128). Thus, under optimal con***ions, the response tends to equalize the large gradient of peptide-MHC complex. Further studies should identify the con***ions that influenced the response. Certainly, the idea of a dominant peptide governing the response is complex (129, 130) and needs to be placed in the context of recent biochemical results.
Finally, we comment on an important phenomenon that we have recently identified as a result of the chemical studies on selected HEL peptides, that of type A and B T cells (119, 131, 132) ( Table 3 , Fig. 8 ). Type A T cells are the conventional T cells induced by immunization with the protein, in our case HEL. Type B T cells, in striking contrast, are induced by immunization with the peptide that has the identical sequence as that resulting from the processing of the protein. These T cells are distinguished because they react with the peptide-pulsed APC but will not react with the APC cultured with the protein ( Table 3 ). In the case of HEL, we can identify and quantitate the major peptide and can confirm the lack of reactivity with the type B cells. The type B, as we have defined them, do not react with post-translational modifications of the peptide (These post-translational modifications can occur with some peptides and will induce specific T cells, but do not explain the A/B reactivity) (133). A very important manipulation in defining the type B reactivity is to isolate the peptide from the class II MHC, after processing of the protein HEL, and to then offer it to APC, this time as an exogenous protein. The type B in such a situation can react to it ( Table 3 )!
In studies in progress by Zheng Pu, Javier Carrero and Daved Fremont, we explain this differential reactivity as caused by a conformational difference in the peptide-MHC complex brought about by the site in the APC where the complex forms ( Fig. 2 ). The type A forms in the deep lysosomal type vesicle under the influence of the catalytic function of H-2 DM; it results in a fixed conformer. In contrast, the type B forms in the early compartment by peptide exchange without the participation of H-2 DM. MHC molecules that peptide-exchange bear weak peptides that have fast dissociation rates; these peptides have side chains that partially occupy the binding pockets and may be more flexible conformationally flexible. Thus, the type B result from more conformation of the peptide with a less constrained MHC molecule (134).
The type B T cells are abundant and have been identified against various peptides of HEL and also against autologous peptides. They represent a substantial number of T cells in the normal repertoire. Peterson and DiPaolo showed that such T cells escape negative selection in HEL transgenic mice (119). We are considering whether, under circumstances of inflammation in tissue where autologous peptides are generated, such T cells can be activated and constitute a component of autoimmunity. Teleologically speaking, the type B T cells should have a function, perhaps to favor interaction with small peptides derived from microbes or to represent a first line of defense in inflammation.

================
Fig. 8. Responses of type A and type B T cells to the peptide 48-61 of HEL. To the left is the response of a type A T cell to HEL and to the 48-61 peptide. The responses are about the same. On the right isthe response of a type B T cell, which recognizes weakly the processed peptide form HEL. Unpublished experiment of Zheng Pu.
=================
Concay

Back to the symbiotic relationship between APC and the lymphocyte
Integrating back to the start of this review, how is the APC system normally functioning in normal con***ions and in the presence of a pathogenic challenge? Normally, the APC system is sampling its environment and processing and presenting autologous peptides. These APC will participate in the thymus in the process of negative and positive selection and in peripheral lymphoid organs. The APC system of the peripheral lymphoid organs is in a quiet and dormant stage that changes into an activation mode by the encounter with a pathogenic organism. As the information on antigen presentation events enlarges, it becomes apparent that the recruitment of T cells and their activation depend not only on the presentation of the peptide-MHC complex but also include various other molecules. Among these are cytokines and chemokines, which are released, costimulatory molecules, as well as molecules involved in cell-to-cell adhesion, expressed on the plasma membrane.
The concept of symbiosis (1) implies that optimal function of cellular immunity requires both the innate system with its APC and the T cells (The innate system comprises not only the APC and phagocytic system but also the natural killer (NK) cells and the various granulocytes that operate early in the response to pathogens). We questioned how the 'innate' system would function in the absence of lymphocytes by examining in detail the response of the severe combined immunodeficiency disease (SCID) mouse to infection with the intracellular pathogen Listeria monocytogenes. The SCID and the RAG mice are devoid of lymphocytes, as a result of a defect in the enzymes that generate the antigen receptors. How autonomous is the innate system, particularly the macrophage system, in responding to pathogens, and what events were lacking in the absence of B and T cells were the questions. The macrophages from SCID mice infected with Listeria produced a series of early cytokines among them interleukin (IL)-1 alpha and beta, IL-12, tumor necrosis factor (TNF) and, as more recently noted, IL-18 (reviewed in 135). Each of these cytokines resulted in the activation of particular cells and triggered inflammatory reactions that resulted in a partial control of the infection. In the absence of T cells, macrophages from SCID mice up-regulated their expression of class II MHC molecules because of interferon-gamma made by the NK cells, and served as potential APC. These events resulted in the activation of the macrophage to a heightened microbicidal function. But, the infected mice would never clear the infection. T cells were required to see the antigen and to activate the inflammatory response that eliminated the pathogen.
The experiments in the SCID mice identified for the first time the extent to which the phagocytes and the innate system can function independently of T cells. They also determined that the innate system cannot operate successfully by itself. But, as we have analyzed here, the adaptive system can not function independently either, particularly at the level of the T-cell system, hence the term symbiosis to characterize their interactions. Recent studies indicate how the APC and other cells of the innate system respond early to pathogens and are activated by way of the Toll-like molecules (136). These studies have added an incredible dimension to explaining the recognition of microbes and will add to our understanding of presentation.


Acknowledgments
We dedicate this review to Ita Askonas who started us in the pathway of research in antigen presentation. I thank present and past colleagues who participated in many facets of our research. And, I emphasize my deep appreciation to my present laboratory group and to Paul Allen, Daved Fremont, Michael Gross and Osami Kanagawa.

References

1. Unanue ER. The regulatory role of macrophages in antigenic stimulation. Part two. symbiotic relationship between lymphocytes and macrophages. Adv Immunol 1981;31: 1-136.

2. Metchnikoff E. Lectures on the Comparative Pathology of Inflammation. New York: Dover Publications, 1968.

3. Ehrlich P. The Croonian lecture. on immunity with special reference to cell life. Proc Royal Soc Lond B Biol Sci 1900;66: 424-431.

4. Koch R. Fortsetzung der Mitteilungen uber ein Heilmittel gegen Tuberkulose. Dtsch Med Wochenstr 1891;9: 101-108.

5. Lurie MB. Resistance to Tuberculosis: Experimental Studies in Native and Acquired Defensive Mechanisms. Cambridge, MA: Harvard Press, 1964.

6. Mackaness GB, Blanden RV. Cellular immunity. Prog Allergy 1967;11: 89-98.

7. Chase MW. The cellular transfer of cutaneous hypersensitivity to tuberculin. Proc Soc Exp Biol Med 1945;59: 34-145.

8. Burnet FM. Modification of Jerne's theory of antibody production using the concept of clonal selection. Aust J Sci 1957;20: 67-74.

9. Miller JFAP. Immunological function of the thymus. Lancet 1961;ii: 748-751.

10. Mitchell GM, Miller JFAP. Cell to cell interaction in the immune response. II. The source of hemolysin-forming cells in irradiated mice given bone marrow and thymus or thoracic duct lymphocytes. J Exp Med 1968;128: 821-832.

11. Davies AJS, Leuchars E, Wallis V, Marchant R, Elliott EV. The failure of thymus-derived cells to produce antibody. Transplantation 1967;5: 222-228.

12. Claman HN, Chaperon EA, Triplett RF. Thymus-marrow cell combinations. Synergism in antibody production. Proc Soc Exp Biol Med 1966;122: 1167-1174.

13. Siskind G, Benacerraf B. Cell selection by antigen in the immune response. Adv Immunol 1969;10: 1-10.

14. Eisen HN, Siskind WG. Variations in affinities of antibodies during the immune response. Biochemistry 1964;3: 996-1002.

15. Naor D, Sulitzeanu D. Binding of radioiodinated bovine serum albumin to mouse spleen cells. Nature 1967;214: 687-688.

16. Sela M. Antigenicity: some molecular aspects. Science 1969;166: 1365-1370.

17. Hedrick SM, Nielsen EA, Kavaler J, Cohen DI, Davis MM. Sequence relationships between putative T-cell receptor polypeptides and immunoglobulins. Nature 1984;308: 153-158.

18. Chien Y, Becker DM, Lindsten T, Okamura M, Cohen DI, Davis MM. A third type of murine T-cell receptor gene. Nature 1984;312: 31-35.

19. Saito H, Kranz DM, Takagaki Y, Hayday AC, Eisen HN, Tonegawa S. A third rearranged and expressed gene in a clone of cytotoxic T lymphocytes. Nature 1984;312: 36-40.

20. McDevitt HO, Sela M. Genetic control of the antibody response. I. Demonstration of determinant-specific differences in response to synthetic polypeptide antigens in two strains of inbred mice. J Exp Med 1965;122: 517-524.

21. Levine BB, Ojeda A, Benacerraf B. Studies on artificial antigens. III. The genetic control of the immune response to hapten poly-L-lysine conjugates in guinea pigs. J Exp Med 1963;118: 953-958.

22. Gell PGH, Benacerraf B. Studies on hypersensitivity to denatured proteins in guinea pigs. Immunology 1959;2: 64-70.

23. Mitchison NA. The carrier effect in the secondary response to hapten-protein conjugates. II. Cellular cooperation. Eur J Immunol 1971;1: 18-24.

24. Shirrmacher V, Wigzell H. Immune responses against native and chemically modified albumins in mice. J Immunol 1974;113: 1635-1643.

25. Ishizaka K, Kishimoto T, Delesperse G, King TP. Presence of specific determinants for T cell in denatured antigen and polypeptide chains. J Immunol 1974;113: 70-74.

26. Chesnut RW, Endres RO, Grey HM. Antigen recognition by T cells and B cells: recognition of cross-reactivity between native and denatured forms of globular antigens. Clin Immunol Immunopathol 1980;15: 297-408.

27. McDevitt HO, Chinitz A. Genetic control of the antibody response. relationship between immune response and histocompatibility (H-2) type. Science 1969;163: 1207-1208.

28. Benacerraf B. Role of MHC gene products in immune regulation. Science 1981;212: 229-238.

29. Zinkernagel RM, Doherty PC. Activity of sensitized thymus-derived lymphocytes in lymphocytic choriomeningitis reflects immunological surveillance against altered self components. Nature 1974;251: 230-233.

30. Katz DH, Hamaoka T, Dorf ME, Maurer PH, Benacerraf B. Cell interactions between histoincompatible T and B lymphocytes. IV. Involvement of immune response (Ir) gene control of lymphocyte interaction controlled by the gene. J Exp Med 1973;138: 734-740.

31. Kindred B, Shreffler DC. H-2 dependence of co-operation between T and B cells in vivo. J Immunol 1972;109: 940-943.

Concay

Back to the symbiotic relationship between APC and the lymphocyte
Integrating back to the start of this review, how is the APC system normally functioning in normal con***ions and in the presence of a pathogenic challenge? Normally, the APC system is sampling its environment and processing and presenting autologous peptides. These APC will participate in the thymus in the process of negative and positive selection and in peripheral lymphoid organs. The APC system of the peripheral lymphoid organs is in a quiet and dormant stage that changes into an activation mode by the encounter with a pathogenic organism. As the information on antigen presentation events enlarges, it becomes apparent that the recruitment of T cells and their activation depend not only on the presentation of the peptide-MHC complex but also include various other molecules. Among these are cytokines and chemokines, which are released, costimulatory molecules, as well as molecules involved in cell-to-cell adhesion, expressed on the plasma membrane.
The concept of symbiosis (1) implies that optimal function of cellular immunity requires both the innate system with its APC and the T cells (The innate system comprises not only the APC and phagocytic system but also the natural killer (NK) cells and the various granulocytes that operate early in the response to pathogens). We questioned how the 'innate' system would function in the absence of lymphocytes by examining in detail the response of the severe combined immunodeficiency disease (SCID) mouse to infection with the intracellular pathogen Listeria monocytogenes. The SCID and the RAG mice are devoid of lymphocytes, as a result of a defect in the enzymes that generate the antigen receptors. How autonomous is the innate system, particularly the macrophage system, in responding to pathogens, and what events were lacking in the absence of B and T cells were the questions. The macrophages from SCID mice infected with Listeria produced a series of early cytokines among them interleukin (IL)-1 alpha and beta, IL-12, tumor necrosis factor (TNF) and, as more recently noted, IL-18 (reviewed in 135). Each of these cytokines resulted in the activation of particular cells and triggered inflammatory reactions that resulted in a partial control of the infection. In the absence of T cells, macrophages from SCID mice up-regulated their expression of class II MHC molecules because of interferon-gamma made by the NK cells, and served as potential APC. These events resulted in the activation of the macrophage to a heightened microbicidal function. But, the infected mice would never clear the infection. T cells were required to see the antigen and to activate the inflammatory response that eliminated the pathogen.
The experiments in the SCID mice identified for the first time the extent to which the phagocytes and the innate system can function independently of T cells. They also determined that the innate system cannot operate successfully by itself. But, as we have analyzed here, the adaptive system can not function independently either, particularly at the level of the T-cell system, hence the term symbiosis to characterize their interactions. Recent studies indicate how the APC and other cells of the innate system respond early to pathogens and are activated by way of the Toll-like molecules (136). These studies have added an incredible dimension to explaining the recognition of microbes and will add to our understanding of presentation.


Acknowledgments
We dedicate this review to Ita Askonas who started us in the pathway of research in antigen presentation. I thank present and past colleagues who participated in many facets of our research. And, I emphasize my deep appreciation to my present laboratory group and to Paul Allen, Daved Fremont, Michael Gross and Osami Kanagawa.

References

1. Unanue ER. The regulatory role of macrophages in antigenic stimulation. Part two. symbiotic relationship between lymphocytes and macrophages. Adv Immunol 1981;31: 1-136.

2. Metchnikoff E. Lectures on the Comparative Pathology of Inflammation. New York: Dover Publications, 1968.

3. Ehrlich P. The Croonian lecture. on immunity with special reference to cell life. Proc Royal Soc Lond B Biol Sci 1900;66: 424-431.

4. Koch R. Fortsetzung der Mitteilungen uber ein Heilmittel gegen Tuberkulose. Dtsch Med Wochenstr 1891;9: 101-108.

5. Lurie MB. Resistance to Tuberculosis: Experimental Studies in Native and Acquired Defensive Mechanisms. Cambridge, MA: Harvard Press, 1964.

6. Mackaness GB, Blanden RV. Cellular immunity. Prog Allergy 1967;11: 89-98.

7. Chase MW. The cellular transfer of cutaneous hypersensitivity to tuberculin. Proc Soc Exp Biol Med 1945;59: 34-145.

8. Burnet FM. Modification of Jerne's theory of antibody production using the concept of clonal selection. Aust J Sci 1957;20: 67-74.

9. Miller JFAP. Immunological function of the thymus. Lancet 1961;ii: 748-751.

10. Mitchell GM, Miller JFAP. Cell to cell interaction in the immune response. II. The source of hemolysin-forming cells in irradiated mice given bone marrow and thymus or thoracic duct lymphocytes. J Exp Med 1968;128: 821-832.

11. Davies AJS, Leuchars E, Wallis V, Marchant R, Elliott EV. The failure of thymus-derived cells to produce antibody. Transplantation 1967;5: 222-228.

12. Claman HN, Chaperon EA, Triplett RF. Thymus-marrow cell combinations. Synergism in antibody production. Proc Soc Exp Biol Med 1966;122: 1167-1174.

13. Siskind G, Benacerraf B. Cell selection by antigen in the immune response. Adv Immunol 1969;10: 1-10.

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31. Kindred B, Shreffler DC. H-2 dependence of co-operation between T and B cells in vivo. J Immunol 1972;109: 940-943.

Concay

32. Rosenthal AS, Shevach EM. Function of macrophages in antigen recognition by guinea pig T lymphocytes. I. Requirement for histocompatible macrophages and lymphocytes. J Exp Med 1973;138: 1194-1212.

33. Klein J. Immunology the Science of Self-Nonself Discrimination. New York: John Wiley & Sons, 1982: 267.

34. Rosenthal AS, Barcinski MA, Blake JT. Determinant selection is a macrophage dependent immune response gene function. Nature 1997;267: 156-158.

35. Dresser DW. Acquired immunological tolerance to a fraction of bovine gamma globulin. Immunology 1961;4: 13-22.

36. Chiller JM, Weigle WO. Biography of a tolerant state: cellular parameters of the unresponsive state induced in adult mice to human gamma globulin. J Reticuloendothel Soc 1975;17: 180-186.

37. Askonas BA, Auzins I, Unanue ER. Role of macrophages in the immune response. Bull Soc Chim Biol 1968;50: 1113-1128.

38. Unanue ER, Askonas BA. The immune response of mice to antigen in macrophages. Immunology 1968;15: 287-296.

39. Unanue ER, Askonas BA. Persistence of immunogenicity of antigen after uptake by macrophages. J Exp Med 1968;127: 915-926.

40. Ziegler K, Unanue E. The specific binding of Listeria monocytogenes-immune T lymphocytes to macrophages. I. Quantitation and role of H-2 gene products. J Exp Med 1979;150: 1143-1160.

41. Werdelin O, Shevach EM. Role of nominal antigen and Ia antigen in the binding of antigen-specific T lymphocytes to macrophages. J Immunol 1979;123: 2779-2784.

42. Inaba K, Steinman RM, Van Voohis WC, Muramatsu S. Dendritic cells are crucial accessory cells for thymus-dependent antibody responses in mouse and man. Proc Natl Acad Sci USA 1983;80: 6041-6045.

43. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392: 245-252.

44. Chesnut RW, Colon SM, Grey HM. Requirements for the processing of antigens by antigen-presenting B cells. I. Functional comparison of B cell tumors and macrophages. J Immunol 1982;129: 2382-2388.

45. Ziegler K, Unanue ER. Identification of a macrophage antigen-processing event required for I-region-restricted antigen presentation to T lymphocytes. J Immunol 1981;127: 1869-1875.

46. Ziegler K, Unanue ER. Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to T cells. Proc Natl Acad Sci USA 1982;79: 175-178.

47. Allen PM, Unanue ER. Differential requirements for antigen processing by macrophages for lysozyme-specific T cell hybridoma. J Immunol 1984;132: 1077-1079.

48. Allen PM, Strydom DJ, Unanue ER. Processing of lysozyme by macrophages: identification of the determinant recognized by two T cell hybridomas. Proc Natl Acad Sci USA 1984;81: 2489-2493.

49. Katz ME, Maizels RM, Wicker L, Miller A, Sercarz EE. Immunological focusing by th mouse major histocompatibility comlex: mouse strains confronted with distantly related lysozymes confine their attention to very few epitopes. Eur J Med 1982;12: 535-540.

50. Humphrey JH. Serendipity in immunology. Annu Rev Immunol 1984;2: 1-21.

51. Shimonkevitz R, Kappler J, Marrack P, Grey H. Antigen recognition by H-2-restricted T cells. I. Cell-free antigen processing. J Exp Med 1983;158: 303-316.

52. Allen PM, Beller DI, Braun J, Unanue ER. The handling of Listeria monocytogenes by macrophages: the search for an immunogenic molecule in antigen presentation. J Immunol 1984;132: 323-331.

53. Nelson CA, Roof RW, McCourt DW, Unanue ER. Identification of the naturally processed form of hen egg white lysozyme bound to the murine major histocompatibility complex class II molecule I-Ak. Proc Natl Acad Sci USA 1992;89: 7380-7383.

54. Babbitt BP, Allen PM, Matsueda G, Haber E, Unanue ER. Binding of immunogenic peptides to Ia histocompatibilty molecules. Nature 1985;317: 359-361.

55. Babbitt BP, Matsueda G, Haber E, Unanue ER, Allen PM. Antigenic competition at the level of peptide-Ia binding. Proc Natl Acad Sci USA 1986;83: 4509-4513.

56. Donermeyer DL, Allen PM. Binding to Ia protects an immunogenic peptide from proteolytic degradation. J Immunol 1989;142: 1063-1068.

57. Carrasco-Marin E, Petzold S, Unanue ER. Two structural states of complexes or peptide and class II major histocompatibility complex revealed by photoaffinity-labeled peptides. J Biol Chem 1999;274: 31333-31340.

58. Germain RN, Hendrix LR. MHC class II structure, occupancy and surface expression determined by post-endoplasmic reticulum antigen binding. Nature 1991;12: 134-139.

59. Germain RN, Rinker AG Jr. Peptide binding inhibits protein aggregation of invariant-chain free class II dimers and promotes surface expression of occupied molecules. Nature 1993;24: 725-728.

60. Buus S, Colon S, Smith C, Freed JH, Miles C, Grey HM. Interaction between a 'processed' ovalbumin peptide and Ia molecules. Proc Natl Acad Sci USA 1986;83: 3968-3971.

61. Buus S, Sette A, Colon SM, Miles C, Grey HM. The relationship between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides. Science 1987;235: 1353-1358.

62. Townsend ARM, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 1986;44: 959-968.

63. Bjorkman PJ, Saper B, Samraoui B, Bennett WS, Strominger JL, Wiley DC. The foreign antigen binding site and T cell recognition regions of class I histocompatibility regions. Nature 1987;329: 512-515.

64. Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger WL, Wiley DC. Structure of the human class I histocompatibility antigen HLA-A2. Nature 1987;329: 506-511.

65. Allen PM, Matsueda GR, Evans RJ, Dunbar JB Jr, Marshall GR, Unanue E. Identification of the T-cell and Ia contact residues of a T-cell antigenic epitope. Nature 1987;327: 713-715.

66. Sette A, Buus S, Colon S, Smith JA, Miles C, Grey HM. Structural characteristics of an antigen required for its interaction with Ia and recognition by T cells. Nature 1987;328: 395-399.

67. Fremont DH, Matsumura M, Stura EA, Peterson PA, Wilson IA. Crystal structures of two viral peptides in complex with murine MHC class I H-2Kb. Science 1992;257: 305-317.

68. Madden DR, Gorga JC, Strominger JL, Wiley DC. The structure of HLA-B27 reveals nonamer self-peptides bound in an extended conformation. Nature 1994;353: 321-325.

69. Stern L, Brown G, Jardetzky T, et al. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an antigenic peptide from influenza virus. Nature 1994;368: 215-219.

70. Jardetzky TS, Brown JH, Gorga JC, et al. Three-dimensional structure of a human class II histocompatibility molecule complexed with superantigen. Nature 1994;368: 711-717.

71. Dessen Z, Lawrence CM, Cupo S, Zaller DM, Wiley DC. X-ray crystal structure of HLA-DR4 (DRA*0101 DRB1*0401) complexed with a peptide from human collagen II. Immunity 1997;7: 473-481.

72. Scott CA, Peterson PA, Teyton L, Wilson IA. Crystal structures of two I-Ad-peptide complexes reveal that high affinity can be achieved without large anchor residues. Immunity 1998;8: 319-329.

73. Fremont DH, Monnaie D, Nelson CA, Hendrickson WA, Unanue ER. Crystal structure of I-Ak in complex with a dominant epitope of lysozyme. Immunity 1998;8: 305-317.

74. Corper AL, Stratmann T, Apostolopoulos V, et al. A structural framework for deciphering the link between I-Ag7 and autoimmune diabetes. Science 2000;288: 505-511.

75. Latek RR, et al. Structural basis of peptide binding and presentation by the type 1 diabetes-associated MHC class II molecule of NOD mice. Immunity 2000;12: 699-710.

Concay

32. Rosenthal AS, Shevach EM. Function of macrophages in antigen recognition by guinea pig T lymphocytes. I. Requirement for histocompatible macrophages and lymphocytes. J Exp Med 1973;138: 1194-1212.

33. Klein J. Immunology the Science of Self-Nonself Discrimination. New York: John Wiley & Sons, 1982: 267.

34. Rosenthal AS, Barcinski MA, Blake JT. Determinant selection is a macrophage dependent immune response gene function. Nature 1997;267: 156-158.

35. Dresser DW. Acquired immunological tolerance to a fraction of bovine gamma globulin. Immunology 1961;4: 13-22.

36. Chiller JM, Weigle WO. Biography of a tolerant state: cellular parameters of the unresponsive state induced in adult mice to human gamma globulin. J Reticuloendothel Soc 1975;17: 180-186.

37. Askonas BA, Auzins I, Unanue ER. Role of macrophages in the immune response. Bull Soc Chim Biol 1968;50: 1113-1128.

38. Unanue ER, Askonas BA. The immune response of mice to antigen in macrophages. Immunology 1968;15: 287-296.

39. Unanue ER, Askonas BA. Persistence of immunogenicity of antigen after uptake by macrophages. J Exp Med 1968;127: 915-926.

40. Ziegler K, Unanue E. The specific binding of Listeria monocytogenes-immune T lymphocytes to macrophages. I. Quantitation and role of H-2 gene products. J Exp Med 1979;150: 1143-1160.

41. Werdelin O, Shevach EM. Role of nominal antigen and Ia antigen in the binding of antigen-specific T lymphocytes to macrophages. J Immunol 1979;123: 2779-2784.

42. Inaba K, Steinman RM, Van Voohis WC, Muramatsu S. Dendritic cells are crucial accessory cells for thymus-dependent antibody responses in mouse and man. Proc Natl Acad Sci USA 1983;80: 6041-6045.

43. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392: 245-252.

44. Chesnut RW, Colon SM, Grey HM. Requirements for the processing of antigens by antigen-presenting B cells. I. Functional comparison of B cell tumors and macrophages. J Immunol 1982;129: 2382-2388.

45. Ziegler K, Unanue ER. Identification of a macrophage antigen-processing event required for I-region-restricted antigen presentation to T lymphocytes. J Immunol 1981;127: 1869-1875.

46. Ziegler K, Unanue ER. Decrease in macrophage antigen catabolism caused by ammonia and chloroquine is associated with inhibition of antigen presentation to T cells. Proc Natl Acad Sci USA 1982;79: 175-178.

47. Allen PM, Unanue ER. Differential requirements for antigen processing by macrophages for lysozyme-specific T cell hybridoma. J Immunol 1984;132: 1077-1079.

48. Allen PM, Strydom DJ, Unanue ER. Processing of lysozyme by macrophages: identification of the determinant recognized by two T cell hybridomas. Proc Natl Acad Sci USA 1984;81: 2489-2493.

49. Katz ME, Maizels RM, Wicker L, Miller A, Sercarz EE. Immunological focusing by th mouse major histocompatibility comlex: mouse strains confronted with distantly related lysozymes confine their attention to very few epitopes. Eur J Med 1982;12: 535-540.

50. Humphrey JH. Serendipity in immunology. Annu Rev Immunol 1984;2: 1-21.

51. Shimonkevitz R, Kappler J, Marrack P, Grey H. Antigen recognition by H-2-restricted T cells. I. Cell-free antigen processing. J Exp Med 1983;158: 303-316.

52. Allen PM, Beller DI, Braun J, Unanue ER. The handling of Listeria monocytogenes by macrophages: the search for an immunogenic molecule in antigen presentation. J Immunol 1984;132: 323-331.

53. Nelson CA, Roof RW, McCourt DW, Unanue ER. Identification of the naturally processed form of hen egg white lysozyme bound to the murine major histocompatibility complex class II molecule I-Ak. Proc Natl Acad Sci USA 1992;89: 7380-7383.

54. Babbitt BP, Allen PM, Matsueda G, Haber E, Unanue ER. Binding of immunogenic peptides to Ia histocompatibilty molecules. Nature 1985;317: 359-361.

55. Babbitt BP, Matsueda G, Haber E, Unanue ER, Allen PM. Antigenic competition at the level of peptide-Ia binding. Proc Natl Acad Sci USA 1986;83: 4509-4513.

56. Donermeyer DL, Allen PM. Binding to Ia protects an immunogenic peptide from proteolytic degradation. J Immunol 1989;142: 1063-1068.

57. Carrasco-Marin E, Petzold S, Unanue ER. Two structural states of complexes or peptide and class II major histocompatibility complex revealed by photoaffinity-labeled peptides. J Biol Chem 1999;274: 31333-31340.

58. Germain RN, Hendrix LR. MHC class II structure, occupancy and surface expression determined by post-endoplasmic reticulum antigen binding. Nature 1991;12: 134-139.

59. Germain RN, Rinker AG Jr. Peptide binding inhibits protein aggregation of invariant-chain free class II dimers and promotes surface expression of occupied molecules. Nature 1993;24: 725-728.

60. Buus S, Colon S, Smith C, Freed JH, Miles C, Grey HM. Interaction between a 'processed' ovalbumin peptide and Ia molecules. Proc Natl Acad Sci USA 1986;83: 3968-3971.

61. Buus S, Sette A, Colon SM, Miles C, Grey HM. The relationship between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides. Science 1987;235: 1353-1358.

62. Townsend ARM, Rothbard J, Gotch FM, Bahadur G, Wraith D, McMichael AJ. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 1986;44: 959-968.

63. Bjorkman PJ, Saper B, Samraoui B, Bennett WS, Strominger JL, Wiley DC. The foreign antigen binding site and T cell recognition regions of class I histocompatibility regions. Nature 1987;329: 512-515.

64. Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger WL, Wiley DC. Structure of the human class I histocompatibility antigen HLA-A2. Nature 1987;329: 506-511.

65. Allen PM, Matsueda GR, Evans RJ, Dunbar JB Jr, Marshall GR, Unanue E. Identification of the T-cell and Ia contact residues of a T-cell antigenic epitope. Nature 1987;327: 713-715.

66. Sette A, Buus S, Colon S, Smith JA, Miles C, Grey HM. Structural characteristics of an antigen required for its interaction with Ia and recognition by T cells. Nature 1987;328: 395-399.

67. Fremont DH, Matsumura M, Stura EA, Peterson PA, Wilson IA. Crystal structures of two viral peptides in complex with murine MHC class I H-2Kb. Science 1992;257: 305-317.

68. Madden DR, Gorga JC, Strominger JL, Wiley DC. The structure of HLA-B27 reveals nonamer self-peptides bound in an extended conformation. Nature 1994;353: 321-325.

69. Stern L, Brown G, Jardetzky T, et al. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an antigenic peptide from influenza virus. Nature 1994;368: 215-219.

70. Jardetzky TS, Brown JH, Gorga JC, et al. Three-dimensional structure of a human class II histocompatibility molecule complexed with superantigen. Nature 1994;368: 711-717.

71. Dessen Z, Lawrence CM, Cupo S, Zaller DM, Wiley DC. X-ray crystal structure of HLA-DR4 (DRA*0101 DRB1*0401) complexed with a peptide from human collagen II. Immunity 1997;7: 473-481.

72. Scott CA, Peterson PA, Teyton L, Wilson IA. Crystal structures of two I-Ad-peptide complexes reveal that high affinity can be achieved without large anchor residues. Immunity 1998;8: 319-329.

73. Fremont DH, Monnaie D, Nelson CA, Hendrickson WA, Unanue ER. Crystal structure of I-Ak in complex with a dominant epitope of lysozyme. Immunity 1998;8: 305-317.

74. Corper AL, Stratmann T, Apostolopoulos V, et al. A structural framework for deciphering the link between I-Ag7 and autoimmune diabetes. Science 2000;288: 505-511.

75. Latek RR, et al. Structural basis of peptide binding and presentation by the type 1 diabetes-associated MHC class II molecule of NOD mice. Immunity 2000;12: 699-710.

Concay

76. Garcia KC. Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. Science 1998;279: 1166-1172.

77. Garboczi DN, Ghosh P, Utz U, Fan QR, Biddison WE, Wiley DC. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature 1996;384: 134-141.

78. Reinherz EL et al. The crystal structure of a T cell receptor in complex with peptide and MHC class II. Science 1999;286: 1913-1921.

79. Hennecke J, Wiley DC. T cell receptor-MHC interactions up close. Cell 2001;104: 1-4.

80. Cresswell P. Assembly, transport, and function of MHC class II molecules. Ann Rev Immunol 1994;12: 259-293.

81. Hudson AW, Ploegh HL. The cell biology of antigen presentation. Exp Cell Res 2002;272: 1-7.

82. Harding CV, Collins DS, Kanagawa O, Unanue ER. Liposome-encapsulated antigens engender lysosomal processing for class II MHC presentation and cytosolic processing for class I presentation. J Immunol 1991;147: 2860-2863.

83. Harding CV, Collins DS, Slot JW, Geuze HJ, Unanue ER. Liposome-encapsulated antigens are processed in lysosomes, recycled, and presented to T cells. Cell 1991;64: 393-401.

84. Phan UT, Arunachalam B, Cresswell P. Gamma-interferon-inducible lysosomal thiol reductase (GILT). Maturation, activity, and mechanism of action. J Biol Chem 2000;275: 25907-25914.

85. Maric M et al. Defective antigen processing in GILT-free mice. Science 2001;294: 1361-1365.

86. Neefjes JJ, Stollorz V, Peters PJ, Geuze HJ, Ploegh HL. The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route. Cell 1990;6: 171-183.

87. Lindner R, Unanue ER. Distinct antigen MHC class II complexes generated by separate processing pathways. EMBO J 1996;15: 6910-6920.

88. Falk K, Rảtzschke O, Stevanovic S, Jung G, Rammensee HG. Pool sequencing of natural HLA-DR, DQ, and DP ligands reveals detailed peptide motifs, constraints of processing, and general rules. Immunogenetics 1994;39: 230-242.

89. Rammensee HG, Friede T, Stevanovic S. MHC ligands and peptide motifs: first listing. Immunogenetics 1995;41: 178-228.

90. Rudensky AY, Preston-Hurlburt P, Hong SC, Barlow A, Janeway JCA. Sequence analysis of peptides bound to MHC class II molecules. Nature 1991;353: 622-627.

91. Chicz RM, et al. Predominant naturally processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size. Nature 1992;358: 764-768.

92. Hunt DF et al. Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I-Ad. Science 1992;256: 1817-1820.

93. Vignali DA, Urban RG, Chicz RM, Strominger JL. Minute quantities of a single dominant foreign epitope are presented as large nested sets by major histocompatibility complex class II molecules. Eur J Immunol 1993;23: 1602-1609.

94. Nelson CA, Vidavsky I, Viner NJ, Gross ML, Unanue ER. Amino-terminal trimming of peptides for presentation on major histocompatibility complex class II molecules. Proc Natl Acad Sci USA 1997;94: 628-933.

95. Gugasyan R, Vidavsky I, Nelson CA, Gross ML, Unanue ER. Isolation and quantitation of a minor determinant of hen egg white lysozyme bound to I-Ak by using peptide-specific immunoaffinity. J Immunol 1998;161: 6074-6083.

96. Hunt DF et al. Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 1992;255: 1261-1263.

97. Gugasyan RC, et al. Independent selection by I-Ak molecules of two epitopes found in tandem in an extended polypeptide. J Immunol 2000;165: 3206-3213.

98. Velazquez C, Vidavsky I, van der Drift K, Gross ML, Unanue ER. Chemical identification of a low abundant lysozyme peptide family bound to I-Ak histocompatibility molecules. J Biol Chem 2002, in press.

99. Velazquez CR, DiPaolo R, Unanue ER. Quantitation of lysozyme peptides bound to class II MHC molecules indicates very large differences in levels of presentation. J Immunol 2001;166: 5488-5494.

100. Suri A, Vidavsky I, van der Drift K, Kanagawa O, Gross ML, Unanue ER. In APCs; the autologous peptides selected by the diabetogenic I-Ag7 molecule are unique and determined by the amino acid changes in the P9 pocket. J Immunol 2002;168: 1235-1243.

101. Dongre AR, et al. In vivo MHC class II presentation of cytosolic proteins revealed by rapid automated tandem mass spectrometry and functional analyses. Eur J Immunol 2001;31: 1485-1494.

102. Mukherjee P, et al. Efficient presentation of both cytosolic and endogenous transmembrane protein antigens on MHC class II is dependent on cytoplasmic proteolysis. J Immunol 2001;167: 2632-2641.

103. Malnati MS, et al. Processing pathways for presentation of cytosolic antigen to MHC class-II restricted T cells. Nature 1992;357: 702-704.

104. Brooks AG, McCluskey J. Class-II restricted presentation of a hen egg lysozyme determinant derived from endogenous antigen sequestered in the cytoplasm or endoplasmic reticulum of the antigen presenting cells. J Immunol 1993;150: 3690-3697.

105. Nelson CA, Viner NJ, Young SP, Petzold SJ, Unanue ER. A negatively charged anchor residue promotes high affinity binding to the MHC class II molecule I-Ak. J Immunol 1996;157: 755-762.

106. Jardetzky T, Gorga J, Busch R, Rothbard J, Strominger J, Wiley D. Peptide binding to HLA-DR1: a simple polyalanine peptide retains MHC binding. EMBO J 1990;9: 1797-1803.

107. Lambert LE, Unanue ER. Analysis of the interaction of peptide hen egg white lysozyme (34-45) with the I-Ak molecule. J Immunol 1989;143: 802-807.

108. Boehncke WH, et al. The importance of dominant negative effects of amino acid side chain substitution in peptide-MHC molecule interactions and T cell recognition. J Immunol 1993;150: 331-341.

109. Sette A, et al. HLA DR4w4-binding motifs illustrate the biochemical basis of degeneracy and specificity in peptide-DR interactions. J Immunol 1993;151: 3163-3170.

110. Latek RR, Petzold S, Unanue ER. Hindering auxiliary anchors are potent modulators of peptide binding and selection by I-Ak class II molecules. Proc Natl Acad Sci USA 2000;97: 11460-11465.

111. Nelson CA, Petzold SJ, Unanue ER. Peptides determine the lifespan of MHC class II molecules in the antigen-presenting cell. Nature 1994;371: 250-252.

112. Carson RT, Vignali KM, Woodland DL, Vignali DA. T cell receptor recognition of MHC class II-bound peptide flanking residues enhances immunogenicity and results in altered TCR V region usage. Immunity 1997;7: 387-399.

113. Tisch R, McDevitt HO. Insulin-dependent diabetes mellitus. Cell 1996;85: 291-298.

114. Acha-Orbea H, McDevitt HO. The first external domain of the nonobese diabetic mouse class II, I-A beta chain is unique. Proc Natl Acad Sci USA 1987;84: 2435-2441.

115. Morel PA, Dorman JS, Todd JA, McDevitt HO, Trucco M. Aspartic acid at position 57 of the HLA-DQ beta chain protects against type I diabetes: a family study[published erratum appears in Proc Natl Acad Sci USA 1989; 86: 1317]. Proc Natl Acad Sci USA 1988;85: 8111-8115.

Concay

76. Garcia KC. Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. Science 1998;279: 1166-1172.

77. Garboczi DN, Ghosh P, Utz U, Fan QR, Biddison WE, Wiley DC. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature 1996;384: 134-141.

78. Reinherz EL et al. The crystal structure of a T cell receptor in complex with peptide and MHC class II. Science 1999;286: 1913-1921.

79. Hennecke J, Wiley DC. T cell receptor-MHC interactions up close. Cell 2001;104: 1-4.

80. Cresswell P. Assembly, transport, and function of MHC class II molecules. Ann Rev Immunol 1994;12: 259-293.

81. Hudson AW, Ploegh HL. The cell biology of antigen presentation. Exp Cell Res 2002;272: 1-7.

82. Harding CV, Collins DS, Kanagawa O, Unanue ER. Liposome-encapsulated antigens engender lysosomal processing for class II MHC presentation and cytosolic processing for class I presentation. J Immunol 1991;147: 2860-2863.

83. Harding CV, Collins DS, Slot JW, Geuze HJ, Unanue ER. Liposome-encapsulated antigens are processed in lysosomes, recycled, and presented to T cells. Cell 1991;64: 393-401.

84. Phan UT, Arunachalam B, Cresswell P. Gamma-interferon-inducible lysosomal thiol reductase (GILT). Maturation, activity, and mechanism of action. J Biol Chem 2000;275: 25907-25914.

85. Maric M et al. Defective antigen processing in GILT-free mice. Science 2001;294: 1361-1365.

86. Neefjes JJ, Stollorz V, Peters PJ, Geuze HJ, Ploegh HL. The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route. Cell 1990;6: 171-183.

87. Lindner R, Unanue ER. Distinct antigen MHC class II complexes generated by separate processing pathways. EMBO J 1996;15: 6910-6920.

88. Falk K, Rảtzschke O, Stevanovic S, Jung G, Rammensee HG. Pool sequencing of natural HLA-DR, DQ, and DP ligands reveals detailed peptide motifs, constraints of processing, and general rules. Immunogenetics 1994;39: 230-242.

89. Rammensee HG, Friede T, Stevanovic S. MHC ligands and peptide motifs: first listing. Immunogenetics 1995;41: 178-228.

90. Rudensky AY, Preston-Hurlburt P, Hong SC, Barlow A, Janeway JCA. Sequence analysis of peptides bound to MHC class II molecules. Nature 1991;353: 622-627.

91. Chicz RM, et al. Predominant naturally processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size. Nature 1992;358: 764-768.

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