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Retroviral Antibody Binding of the MHC Class II Molecule: A Biochemical Influence on CD4 T Cell Differentiation in HIV Infection?
J. theor. Biol. (2001) 209, 155}160 doi:10.1006/jtbi.2000.2245, available online at http://www.idealibrary.com on
Retroviral Antibody Binding of the MHC Class II Molecule: A Biochemical In6uence on CD4 T Cell Di4erentiation in HIV Infection? P. D. POWELL
AND
J. C. DEMARTINI*-
*Department of Pathology, Colorado State ;niversity, Ft. Collins, CO 80523-1671, ;.S.A. (Received on 25 September 2000, Accepted in revised form on 2 December 2000)
Retroviral antibody capable of binding to the major histocompatibility complex (MHC) Class II molecule has been documented in human immunode"ciency virus-1 (HIV-1)-infected patients. Interactions between the MHC Class II receptor and the T-cell receptor (TCR) are central to the immune response. Importantly, retroviral antibody possesses a much higher binding a$nity for the MHC Class II receptor, when compared to the TCR. Experiments have manipulated a number of factors related to antigen-presenting cell (APC) interaction with di!erentiating T-cells. These studies have observed the e!ects of lowering antigen dose and reducing ligand density on precursor Th (T helper) cell di!erentiation. Studies have also examined the e!ect of downregulated MHC Class II receptors and co-stimulatory molecules on APC}Th cell interaction. In addition, the sequestration of antigens away from the Class II processing pathway has been studied. These investigations reveal a general trend that can determine whether a naive CD4 T-cell becomes a Th1 or Th2-like cell. If the experimental manipulation weakens the APC-Th cell interaction, a weak ligating TCR signal results. Consequently, a weak ligating TCR signal can in#uence precursor Th cells to become Th2-like cells. Retroviral antibody binding of MHC Class II receptors may mimic a number of experimental conditions responsible for creating a weak ligating TCR signal. 2001 Academic Press
MHC Class II Antibody = Immune Response to a Peptide Similarity of HIV-1 Studies performed by several investigators con"rm the presence of retroviral antibody capable of binding to the major histocompatibility complex (MHC) Class II molecule (Golding et al., 1988, 1989; Blackburn et al., 1991; Zaitseva et al., 1992; Brondz et al., 1992). Antibody is generated during the immune response to the HIV-1 transmembrane envelope protein. A "ve-amino-acid sequence similarity exists between the viral envelope protein and the DR Beta MHC Class II molecule (Golding et al., 1988, 1989; Blackburn - Author to whom correspondence should be addressed. E-mail: [email protected] 0022}5193/01/060151#06 $35.00/0
et al., 1991). Based on these studies, it was proposed that retroviral antibody binding of the MHC Class II molecule might be responsible for the early impairment of CD4 T-cell function in HIV infection. Experiments were performed on the MHC Class-II-reactive sera from HIV-positive individuals (Golding et al., 1988, 1989; Blackburn et al., 1991). These experiments determined the potential immune impact of antibody binding to the MHC Class II molecule. Sera containing the MHC Class II antibody did markedly impact several indicators of immune function. Proliferative responses of normal CD4 cells to tetanus toxoid and allogenic stimuli were markedly decreased. In addition, reactive sera could eliminate MHC Class-II-bearing cells by a mechanism 2001 Academic Press
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known as antibody dependent cellular cytotoxicity (ADCC) (Golding et al., 1988, 1989; Blackburn et al., 1991). These experiments, however, did not consider the biochemical consequences of the MHC Class II antibody. Since the original experiments were performed, new information has become available regarding the importance of MHC Class II}TCR interactions during the process of T-cell di!erentiation. The Biochemistry of T-cell=MHC Class II Interactions During a normal immune response, the T-cell receptor and MHC Class II molecules interact physically. The interaction between these two receptors forms a central basis for a number of immune system communications. During the initial stages of the immune response, microbial proteins of the infectious agent are processed into peptides. These bacterial or viral peptides are subsequently presented on the peptide binding groove of the MHC Class II receptor (Fremont et al., 1992; Madden et al., 1992). The MHC class II receptor is present on antigen-presenting cells (APC), such as dendritic cells and macrophages. The T-cell receptor then binds to the MHC Class II receptor}peptide complex, initiating the immune response. Importantly, the binding a$nities of the TCR for the MHC Class II molecule range from approximately (K , 10(-5) to 10(-7) mol/l) (Davis et al., 1998; B Salzmann & Bachmann, 1998; Fremont et al., 1996b; Davis & Chien, 1993). Antibody a$nity for its antigen ranges from (K , 10(-9) to 10(-11) mol/l) B (Friguet et al., 1985; Bator & Reading, 1989). Therefore, retroviral antibody possesses a 100-fold higher a.nity for the MHC Class II molecule, when compared to the ¹-cell receptor. This biochemical fact suggests that retroviral antibody is likely to be a more e!ective molecular competitor for MHC class II molecules, compared to the TCR. Antibody may be able to interfere with TCR}MHC II interactions based upon the biochemical dynamics that determine protein binding. Antibody Binding to MHC: Peptide Complexes during CD4 T-cell Di4erentiation The "rst step in the adaptive immune response is the activation of naive T-cells. This event
occurs in the draining lymphoid organs. First, there is an uptake of antigen by interdigitating reticular cells, also known as dendritic cells. Migrating T-cells scan the surface of these dendritic cells for speci"c peptide : MHC complexes (Ebnet et al., 1996; Roake et al., 1995; Janeway et al., 1999a). If the T-cells do not recognize the antigen presented by these cells, they will leave the lymph node within minutes to hours. However, on rare occasions, a naive T-cell will recognize its speci"c peptide : MHC complex on the surface of the dendritic cell (Ebnet et al., 1996; Roake et al., 1995; Janeway et al., 1999a). When this recognition event occurs, an activation signal is emitted that causes the T-cell to adhere strongly to the dendritic cell (Ebnet et al., 1996). When binding of the peptide : MHC complex occurs, co-stimulatory molecules on the surface of the dendritic cell activate the nam ve T-cell to di!erentiate and proliferate. These events culminate in the production of armed, antigen-speci"c, e!ector T-cells. If retroviral antibody is bound to the MHC Class II molecules of dendritic cells, the dynamics of T-cell di!erentiation may be altered. Naive T-cell recognition of antigen is a rare event under normal circumstances. Is it possible that this event occurs even more rarely in HIV-positive individuals? Antibody bound to a signi"cant number of dendritic cells in the lymphoid tissue could have one important immunological outcome: to further reduce the probability that naive T-cells will recognize their corresponding antigen. The e$ciency with which naive T-cells screen each antigen-presenting cell in the lymph nodes is very high. Rapid trapping of antigen-speci"c naive T-cells has been demonstrated in a single lymph node that contained antigen (Ebnet et al., 1996; Roake et al., 1995; Janeway et al., 1999a). All antigen-speci"c T-cells can be trapped in a lymph node within 48 hrs of antigen deposition (Ebnet et al., 1996; Roake et al., 1995; Janeway et al., 1999a). In HIV-infected individuals, could antibody bound to dendritic cells in#uence the e$ciency of antigen-speci"c T-cell trapping? If a speci"c antigen is being blocked from interacting with the T-cell, the e$ciency of antigenspeci"c T-cell trapping could be reduced. This scenario may partially explain impairments of
ANTIBODY & MHC CLASS II-TCR INTERACTIONS
the adaptive immune response that are observed in HIV infection/AIDS (Fauci et al., 1996; Piconi & Clerici, 1997; Cohen et al., 1997). Survival of Naive CD4 T-cells=Interaction with MHC : Peptide Complexes Interaction with MHC : peptide complexes may be necessary to insure the ongoing survival of naive CD4 T-cells (Picker & Butcher, 1992; Pierre et al., 1997). As naive T-cells migrate through the lymphoid tissues, they receive speci"c survival signals through their interactions with dendritic cells. These interactions are delivered e!ectively through the MHC : peptide complexes present on dendritic cells (Picker & Butcher, 1992; Pierre et al., 1997). Since a number of MHC : peptide complexes are likely to be bound to MHC Class II antibody, the availability of these survival signals to naive CD4 T-cells during HIV infection is questionable. Antigen Dose and CD4 T-cell Di4erentiation The immune response to a microbial or viral protein can be in#uenced by antigen dose. Very high or low doses of antigen expression can result in a state of immune unresponsiveness to the antigen (Janeway et al., 1999b). Low doses of antigen can result in low zone tolerance, and consequently there is no immune response to the low level of antigen. Antibody binding of MHC Class II molecules in the lymphoid tissues might e!ectively lower the amount of antigen available for TCR interaction. Eventually, a low antigen dose of microbial peptides from opportunistic microbes could in#uence the immune response. In theory, normal immune responses to opportunistic pathogens require a su$cient antigen dose in order to be properly stimulated. Naive CD4 T-cell development appears to be in#uenced by antigen dose (Secrist et al., 1995; Constant et al., 1995a; Constant & Bottomly, 1997; Hosken et al., 1995). Several studies have demonstrated a selective activation of Th2 immune responses in CD4 T-cells during conditions of low antigen dose (Constant et al., 1995a; Constant & Bottomly, 1997; Wang et al., 1996; Hosken et al., 1995). Reducing the overall number of antigenic complexes available for
157
TCR binding can promote the Th2 di!erentiation of naive CD4 T-cells (Constant & Bottomly, 1997). In studies involving the mouse model, antigen complexes were reduced by altering antigen dose or antigen a$nity for the MHC Class II molecule. Subsequently, naive CD4 T-cells were primed to di!erentiate into Th2-like cells (Constant et al., 1995a; Constant & Bottomly, 1997; Leitenberg et al., 1998). The concept of antigen dose or antigen load is controversial. Determinations of high vs. low antigen load can be subjective in nature. Certain investigators feel that a medium-load level exists as well. During bench experiments, the type of antigen (mitogen, non-speci"c, speci"c) employed to stimulate immune cells is also very important in determining antigen load. Naive CD4 T-cells are probably activated at the site of infection and in the neighboring lymph nodes. Based on the available data, it is generally assumed that naive CD4 T cells are "rst activated into a Th0 state (Kaufmann & Doherty, 1997). These Th0 cells serve as precursor cells for Th1 and Th2 cells (Kaufmann & Doherty, 1997). Investigators have determined that CD4 T-cell di!erentiation can be in#uenced by a number of factors unrelated to antigen dose. These factors are the strength of signals that induce co-stimulation (Gause et al., 1997), the nature of the peptide}ligand interaction (Boutin et al., 1997), the MHC Class II binding a$nity, including receptor}ligand complexes and timing (DiMolfetto et al., 1998), the type and concentration of antigen (Constant et al., 1995b), and the cytokine environment at the time of T-cell priming and activation (Miner & Croft, 1998). Since there are many factors involved in determining T-cell di!erentiation, there is a question regarding the relative importance of antibody binding to the MHC Class II molecule. Speci"cally, the extent of antibody binding to MHC Class II : peptide complexes in the lymphoid tissue of HIV-infected individuals is unknown. This question is important because antibody binding could play a role in the T-cell di!erentiation process of HIV-infected individuals. However, many investigators believe that abnormal T-cell di!erentiation in the form of a Th1 to Th2 shift (Clerici & Shearer, 1993, 1994) may not exist, or exists only in a small subset of HIV-infected individuals.
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Other Factors Determining Th Cell Di4erentiation One study illustrates a critical factor in determining Th cell di!erentiation (Pfei!er et al., 1991). This factor is the number of antigenic epitopes ultimately made available to the antigen-speci"c precursor Th cell (Pfei!er et al., 1991; Constant & Bottomly, 1997). The number of antigenic epitopes, also known as the ligand density, represents the &&binding environment'' of the TCR during the process of T-cell priming. If the ligand density is reduced, this change in the TCR binding environment may bias the process of Th cell development. It is believed that T-cell di!erentiation favors the development of Th2-like cells in a binding environment where the number of antigenic epitopes is reduced (Constant & Bottomly, 1997). Experiments demonstrating a total reduction in the number of antigenic epitopes available for TCR binding could be important. These studies may parallel the in vivo conditions of Th cell di!erentiation that occur in the lymphoid tissue of HIV-positive individuals. The number of antigenic epitopes available for TCR binding could be reduced in seropositive individuals. Several other experimental determinations appear to resemble the functional outcome of retroviral antibody bound to the MHC Class II receptor. Downregulation of MHC Class II receptors or costimulatory molecules can reduce or weaken APC}TCR interactions (Saha et al., 1995; Gupta et al., 1996). In addition, it has been shown that sequestration of antigens away from the Class II processing pathway can also weaken interactions between the APC and the precursor Th cell (Kima et al., 1996; Leyva-Cobian & Unanue, 1988; Prina et al., 1996). All three of these circumstances may alter Th cell development, possibly selecting for Th2-like cells (Constant & Bottomly, 1997).
Conclusion Host immune responses to the infecting virus may contribute to the decline of cell-mediated immunity in HIV infection. The immune response to HIV-1 results in the production of antibody capable of binding to the MHC Class II molecule. Does this immune response contribute
to the major disease consequences observed in HIV infection/AIDS? Fortunately, a small portion of the retroviral genome is responsible for eliciting the potentially detrimental antibody response. It is possible to know the impact of this immune response on overall immune functioning. For instance, in HIV-1-infected individuals progressing to AIDS, the following experiment can be performed. The suspect retroviral sequence, GTDRV can be substituted with the amino acid alanine, AAAAA. Utilizing site-directed mutagenesis on a viral clone of HIV-1, the sequence responsible for eliciting antibodies to the MHC Class II molecule can be replaced. Since the amino acid substitutions are not radical, there will hopefully be no impact on viral protein function. Antibodies generated to this new segment of the retroviral peptide will not recognize the MHC Class II peptide, which is GTERV. The next step is to create a genetic opportunity for the viral clone to establish itself in the quasi-species of the infected host. This can be accomplished by the administration of antiviral medication to the infected host. As a result of reducing viral load, the quasi-species will become genetically unstable. This circumstance then permits the alanine altered clone to be introduced in large numbers to the host. In addition to the alanine substitution, the viral clone will also be engineered to possess anti-viral drug resistance to the medication being administered to the host. As a result of this intervention, the newly reformed quasi-species will contain viruses that possess drug resistance, in addition to the alanine-substitution. Viruses containing the original GTDRV sequence will be suppressed by the presence of the anti-viral medication. Therefore, the AAAAA mutation will become fully established and predominant in the newly reformed quasi-species. Once viral load is signi"cantly raised, the anti-viral medication can then be withdrawn from the host. The measurement of immunological parameters can be utilized to determine the impact of this intervention. This approach is based upon the concept of evolutionary stable strategy. It is explained in greater detail in another work (Powell et al., 2000).
ANTIBODY & MHC CLASS II-TCR INTERACTIONS
If antibody production to the suspect sequence is not responsible for initiating the disease process, immune function will begin to deteriorate as a result of increasing viral load. In this event, treatment of the infected individual with a second, and di!erent anti-viral medication, will lower the viral load and temporarily restore immune function. Genetic engineering of viruses has been proposed as a tool in numerous disease models. This tool is being investigated in a number of applications involving gene therapy and vaccine development (Palese, 1998). It may now also be possible to engineer the retroviral genome in order to improve human health. Shaping the genetic content of the infecting retroviral quasi-species could also be a research tool. This tool could be employed to alter a speci"c host immune response to the virus. Eliminating the suspect sequence is equivalent to eliminating speci"c immune responses to the sequence. Consequently, it could be determined whether the original antibody is contributing to the disease process. This method may precisely determine the role of potentially deleterious immune responses on host immune functioning. In the example illustrated, renewed antibody production to the substituted sequence will not result in recognition or binding to the host protein (MHC Class II receptor). Therefore, in the absence of retroviral antibody capable of binding the MHC Class II molecule, proposed contributions of the antibody to disease mechanisms could be determined in a de"nitive manner.
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Retroviral Antibody Binding of the MHC Class II Molecule: A Biochemical In6uence on CD4 T Cell Di4erentiation in HIV Infection? P. D. POWELL
AND
J. C. DEMARTINI*-
*Department of Pathology, Colorado State ;niversity, Ft. Collins, CO 80523-1671, ;.S.A. (Received on 25 September 2000, Accepted in revised form on 2 December 2000)
Retroviral antibody capable of binding to the major histocompatibility complex (MHC) Class II molecule has been documented in human immunode"ciency virus-1 (HIV-1)-infected patients. Interactions between the MHC Class II receptor and the T-cell receptor (TCR) are central to the immune response. Importantly, retroviral antibody possesses a much higher binding a$nity for the MHC Class II receptor, when compared to the TCR. Experiments have manipulated a number of factors related to antigen-presenting cell (APC) interaction with di!erentiating T-cells. These studies have observed the e!ects of lowering antigen dose and reducing ligand density on precursor Th (T helper) cell di!erentiation. Studies have also examined the e!ect of downregulated MHC Class II receptors and co-stimulatory molecules on APC}Th cell interaction. In addition, the sequestration of antigens away from the Class II processing pathway has been studied. These investigations reveal a general trend that can determine whether a naive CD4 T-cell becomes a Th1 or Th2-like cell. If the experimental manipulation weakens the APC-Th cell interaction, a weak ligating TCR signal results. Consequently, a weak ligating TCR signal can in#uence precursor Th cells to become Th2-like cells. Retroviral antibody binding of MHC Class II receptors may mimic a number of experimental conditions responsible for creating a weak ligating TCR signal. 2001 Academic Press
MHC Class II Antibody = Immune Response to a Peptide Similarity of HIV-1 Studies performed by several investigators con"rm the presence of retroviral antibody capable of binding to the major histocompatibility complex (MHC) Class II molecule (Golding et al., 1988, 1989; Blackburn et al., 1991; Zaitseva et al., 1992; Brondz et al., 1992). Antibody is generated during the immune response to the HIV-1 transmembrane envelope protein. A "ve-amino-acid sequence similarity exists between the viral envelope protein and the DR Beta MHC Class II molecule (Golding et al., 1988, 1989; Blackburn - Author to whom correspondence should be addressed. E-mail: [email protected] 0022}5193/01/060151#06 $35.00/0
et al., 1991). Based on these studies, it was proposed that retroviral antibody binding of the MHC Class II molecule might be responsible for the early impairment of CD4 T-cell function in HIV infection. Experiments were performed on the MHC Class-II-reactive sera from HIV-positive individuals (Golding et al., 1988, 1989; Blackburn et al., 1991). These experiments determined the potential immune impact of antibody binding to the MHC Class II molecule. Sera containing the MHC Class II antibody did markedly impact several indicators of immune function. Proliferative responses of normal CD4 cells to tetanus toxoid and allogenic stimuli were markedly decreased. In addition, reactive sera could eliminate MHC Class-II-bearing cells by a mechanism 2001 Academic Press
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known as antibody dependent cellular cytotoxicity (ADCC) (Golding et al., 1988, 1989; Blackburn et al., 1991). These experiments, however, did not consider the biochemical consequences of the MHC Class II antibody. Since the original experiments were performed, new information has become available regarding the importance of MHC Class II}TCR interactions during the process of T-cell di!erentiation. The Biochemistry of T-cell=MHC Class II Interactions During a normal immune response, the T-cell receptor and MHC Class II molecules interact physically. The interaction between these two receptors forms a central basis for a number of immune system communications. During the initial stages of the immune response, microbial proteins of the infectious agent are processed into peptides. These bacterial or viral peptides are subsequently presented on the peptide binding groove of the MHC Class II receptor (Fremont et al., 1992; Madden et al., 1992). The MHC class II receptor is present on antigen-presenting cells (APC), such as dendritic cells and macrophages. The T-cell receptor then binds to the MHC Class II receptor}peptide complex, initiating the immune response. Importantly, the binding a$nities of the TCR for the MHC Class II molecule range from approximately (K , 10(-5) to 10(-7) mol/l) (Davis et al., 1998; B Salzmann & Bachmann, 1998; Fremont et al., 1996b; Davis & Chien, 1993). Antibody a$nity for its antigen ranges from (K , 10(-9) to 10(-11) mol/l) B (Friguet et al., 1985; Bator & Reading, 1989). Therefore, retroviral antibody possesses a 100-fold higher a.nity for the MHC Class II molecule, when compared to the ¹-cell receptor. This biochemical fact suggests that retroviral antibody is likely to be a more e!ective molecular competitor for MHC class II molecules, compared to the TCR. Antibody may be able to interfere with TCR}MHC II interactions based upon the biochemical dynamics that determine protein binding. Antibody Binding to MHC: Peptide Complexes during CD4 T-cell Di4erentiation The "rst step in the adaptive immune response is the activation of naive T-cells. This event
occurs in the draining lymphoid organs. First, there is an uptake of antigen by interdigitating reticular cells, also known as dendritic cells. Migrating T-cells scan the surface of these dendritic cells for speci"c peptide : MHC complexes (Ebnet et al., 1996; Roake et al., 1995; Janeway et al., 1999a). If the T-cells do not recognize the antigen presented by these cells, they will leave the lymph node within minutes to hours. However, on rare occasions, a naive T-cell will recognize its speci"c peptide : MHC complex on the surface of the dendritic cell (Ebnet et al., 1996; Roake et al., 1995; Janeway et al., 1999a). When this recognition event occurs, an activation signal is emitted that causes the T-cell to adhere strongly to the dendritic cell (Ebnet et al., 1996). When binding of the peptide : MHC complex occurs, co-stimulatory molecules on the surface of the dendritic cell activate the nam ve T-cell to di!erentiate and proliferate. These events culminate in the production of armed, antigen-speci"c, e!ector T-cells. If retroviral antibody is bound to the MHC Class II molecules of dendritic cells, the dynamics of T-cell di!erentiation may be altered. Naive T-cell recognition of antigen is a rare event under normal circumstances. Is it possible that this event occurs even more rarely in HIV-positive individuals? Antibody bound to a signi"cant number of dendritic cells in the lymphoid tissue could have one important immunological outcome: to further reduce the probability that naive T-cells will recognize their corresponding antigen. The e$ciency with which naive T-cells screen each antigen-presenting cell in the lymph nodes is very high. Rapid trapping of antigen-speci"c naive T-cells has been demonstrated in a single lymph node that contained antigen (Ebnet et al., 1996; Roake et al., 1995; Janeway et al., 1999a). All antigen-speci"c T-cells can be trapped in a lymph node within 48 hrs of antigen deposition (Ebnet et al., 1996; Roake et al., 1995; Janeway et al., 1999a). In HIV-infected individuals, could antibody bound to dendritic cells in#uence the e$ciency of antigen-speci"c T-cell trapping? If a speci"c antigen is being blocked from interacting with the T-cell, the e$ciency of antigenspeci"c T-cell trapping could be reduced. This scenario may partially explain impairments of
ANTIBODY & MHC CLASS II-TCR INTERACTIONS
the adaptive immune response that are observed in HIV infection/AIDS (Fauci et al., 1996; Piconi & Clerici, 1997; Cohen et al., 1997). Survival of Naive CD4 T-cells=Interaction with MHC : Peptide Complexes Interaction with MHC : peptide complexes may be necessary to insure the ongoing survival of naive CD4 T-cells (Picker & Butcher, 1992; Pierre et al., 1997). As naive T-cells migrate through the lymphoid tissues, they receive speci"c survival signals through their interactions with dendritic cells. These interactions are delivered e!ectively through the MHC : peptide complexes present on dendritic cells (Picker & Butcher, 1992; Pierre et al., 1997). Since a number of MHC : peptide complexes are likely to be bound to MHC Class II antibody, the availability of these survival signals to naive CD4 T-cells during HIV infection is questionable. Antigen Dose and CD4 T-cell Di4erentiation The immune response to a microbial or viral protein can be in#uenced by antigen dose. Very high or low doses of antigen expression can result in a state of immune unresponsiveness to the antigen (Janeway et al., 1999b). Low doses of antigen can result in low zone tolerance, and consequently there is no immune response to the low level of antigen. Antibody binding of MHC Class II molecules in the lymphoid tissues might e!ectively lower the amount of antigen available for TCR interaction. Eventually, a low antigen dose of microbial peptides from opportunistic microbes could in#uence the immune response. In theory, normal immune responses to opportunistic pathogens require a su$cient antigen dose in order to be properly stimulated. Naive CD4 T-cell development appears to be in#uenced by antigen dose (Secrist et al., 1995; Constant et al., 1995a; Constant & Bottomly, 1997; Hosken et al., 1995). Several studies have demonstrated a selective activation of Th2 immune responses in CD4 T-cells during conditions of low antigen dose (Constant et al., 1995a; Constant & Bottomly, 1997; Wang et al., 1996; Hosken et al., 1995). Reducing the overall number of antigenic complexes available for
157
TCR binding can promote the Th2 di!erentiation of naive CD4 T-cells (Constant & Bottomly, 1997). In studies involving the mouse model, antigen complexes were reduced by altering antigen dose or antigen a$nity for the MHC Class II molecule. Subsequently, naive CD4 T-cells were primed to di!erentiate into Th2-like cells (Constant et al., 1995a; Constant & Bottomly, 1997; Leitenberg et al., 1998). The concept of antigen dose or antigen load is controversial. Determinations of high vs. low antigen load can be subjective in nature. Certain investigators feel that a medium-load level exists as well. During bench experiments, the type of antigen (mitogen, non-speci"c, speci"c) employed to stimulate immune cells is also very important in determining antigen load. Naive CD4 T-cells are probably activated at the site of infection and in the neighboring lymph nodes. Based on the available data, it is generally assumed that naive CD4 T cells are "rst activated into a Th0 state (Kaufmann & Doherty, 1997). These Th0 cells serve as precursor cells for Th1 and Th2 cells (Kaufmann & Doherty, 1997). Investigators have determined that CD4 T-cell di!erentiation can be in#uenced by a number of factors unrelated to antigen dose. These factors are the strength of signals that induce co-stimulation (Gause et al., 1997), the nature of the peptide}ligand interaction (Boutin et al., 1997), the MHC Class II binding a$nity, including receptor}ligand complexes and timing (DiMolfetto et al., 1998), the type and concentration of antigen (Constant et al., 1995b), and the cytokine environment at the time of T-cell priming and activation (Miner & Croft, 1998). Since there are many factors involved in determining T-cell di!erentiation, there is a question regarding the relative importance of antibody binding to the MHC Class II molecule. Speci"cally, the extent of antibody binding to MHC Class II : peptide complexes in the lymphoid tissue of HIV-infected individuals is unknown. This question is important because antibody binding could play a role in the T-cell di!erentiation process of HIV-infected individuals. However, many investigators believe that abnormal T-cell di!erentiation in the form of a Th1 to Th2 shift (Clerici & Shearer, 1993, 1994) may not exist, or exists only in a small subset of HIV-infected individuals.
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Other Factors Determining Th Cell Di4erentiation One study illustrates a critical factor in determining Th cell di!erentiation (Pfei!er et al., 1991). This factor is the number of antigenic epitopes ultimately made available to the antigen-speci"c precursor Th cell (Pfei!er et al., 1991; Constant & Bottomly, 1997). The number of antigenic epitopes, also known as the ligand density, represents the &&binding environment'' of the TCR during the process of T-cell priming. If the ligand density is reduced, this change in the TCR binding environment may bias the process of Th cell development. It is believed that T-cell di!erentiation favors the development of Th2-like cells in a binding environment where the number of antigenic epitopes is reduced (Constant & Bottomly, 1997). Experiments demonstrating a total reduction in the number of antigenic epitopes available for TCR binding could be important. These studies may parallel the in vivo conditions of Th cell di!erentiation that occur in the lymphoid tissue of HIV-positive individuals. The number of antigenic epitopes available for TCR binding could be reduced in seropositive individuals. Several other experimental determinations appear to resemble the functional outcome of retroviral antibody bound to the MHC Class II receptor. Downregulation of MHC Class II receptors or costimulatory molecules can reduce or weaken APC}TCR interactions (Saha et al., 1995; Gupta et al., 1996). In addition, it has been shown that sequestration of antigens away from the Class II processing pathway can also weaken interactions between the APC and the precursor Th cell (Kima et al., 1996; Leyva-Cobian & Unanue, 1988; Prina et al., 1996). All three of these circumstances may alter Th cell development, possibly selecting for Th2-like cells (Constant & Bottomly, 1997).
Conclusion Host immune responses to the infecting virus may contribute to the decline of cell-mediated immunity in HIV infection. The immune response to HIV-1 results in the production of antibody capable of binding to the MHC Class II molecule. Does this immune response contribute
to the major disease consequences observed in HIV infection/AIDS? Fortunately, a small portion of the retroviral genome is responsible for eliciting the potentially detrimental antibody response. It is possible to know the impact of this immune response on overall immune functioning. For instance, in HIV-1-infected individuals progressing to AIDS, the following experiment can be performed. The suspect retroviral sequence, GTDRV can be substituted with the amino acid alanine, AAAAA. Utilizing site-directed mutagenesis on a viral clone of HIV-1, the sequence responsible for eliciting antibodies to the MHC Class II molecule can be replaced. Since the amino acid substitutions are not radical, there will hopefully be no impact on viral protein function. Antibodies generated to this new segment of the retroviral peptide will not recognize the MHC Class II peptide, which is GTERV. The next step is to create a genetic opportunity for the viral clone to establish itself in the quasi-species of the infected host. This can be accomplished by the administration of antiviral medication to the infected host. As a result of reducing viral load, the quasi-species will become genetically unstable. This circumstance then permits the alanine altered clone to be introduced in large numbers to the host. In addition to the alanine substitution, the viral clone will also be engineered to possess anti-viral drug resistance to the medication being administered to the host. As a result of this intervention, the newly reformed quasi-species will contain viruses that possess drug resistance, in addition to the alanine-substitution. Viruses containing the original GTDRV sequence will be suppressed by the presence of the anti-viral medication. Therefore, the AAAAA mutation will become fully established and predominant in the newly reformed quasi-species. Once viral load is signi"cantly raised, the anti-viral medication can then be withdrawn from the host. The measurement of immunological parameters can be utilized to determine the impact of this intervention. This approach is based upon the concept of evolutionary stable strategy. It is explained in greater detail in another work (Powell et al., 2000).
ANTIBODY & MHC CLASS II-TCR INTERACTIONS
If antibody production to the suspect sequence is not responsible for initiating the disease process, immune function will begin to deteriorate as a result of increasing viral load. In this event, treatment of the infected individual with a second, and di!erent anti-viral medication, will lower the viral load and temporarily restore immune function. Genetic engineering of viruses has been proposed as a tool in numerous disease models. This tool is being investigated in a number of applications involving gene therapy and vaccine development (Palese, 1998). It may now also be possible to engineer the retroviral genome in order to improve human health. Shaping the genetic content of the infecting retroviral quasi-species could also be a research tool. This tool could be employed to alter a speci"c host immune response to the virus. Eliminating the suspect sequence is equivalent to eliminating speci"c immune responses to the sequence. Consequently, it could be determined whether the original antibody is contributing to the disease process. This method may precisely determine the role of potentially deleterious immune responses on host immune functioning. In the example illustrated, renewed antibody production to the substituted sequence will not result in recognition or binding to the host protein (MHC Class II receptor). Therefore, in the absence of retroviral antibody capable of binding the MHC Class II molecule, proposed contributions of the antibody to disease mechanisms could be determined in a de"nitive manner.
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