The cellular immune system is the main component of adaptive immune responses that target virally infected cells and is preferentially destroyed in HIV disease. This section will highlight aspects of innate and adaptive immunity including T-cell development, antigen presentation and recognition, T-cell memory, lymph node architecture and chemokine biology relevant to HIV disease pathogenesis.
Stem cells continuously migrate from the bone marrow to the thymus. Although the thymus partially involutes with age, T cells continue to develop in the thymus throughout life. T-cell development in the thymus involves processes for both positive and negative selection that result in the production of T cells with the capacity to recognise foreign or non-self antigens (Ag) (Figure 1). In humanised mouse models of HIV infection, reconstitution of the human immune depends on thymic reconstitution or transplantation. The relationship between thymic production and subsequent destruction of T cells is summarised in Figure 2.
Note: The thymus is divided into lobules separated by trabeculae. Each lobule has a cortex and medulla. T-cell precursors enter the thymus and conditioned naïve T cells exit at the corticomedullary junction (a). The immature double negative (DN; CD3-CD4-CD8-) thymocytes develop and migrate to the cortex where they interact with plasmacytoid dendritic cells (pDC) and cortical thymic epithelial cells (cTEC) (b). Positive selection for low affinity T-cell receptors interacting with self major histocompatibility complex (MHC) molecules and expansion of double positive (DP; CD3+CD4+CD8+) cortical thymocytes occurs in the cortex (c). The positively selected single positive (SP; CD3+CD4+CD8- and CD3+CD4-CD8+) thymocytes migrate to the corticomedullary junction where further interactions with myeloid dendritic cells (mDC) and pDC and medullary thymic epithelial cells (mTEC) lead to negative selection and elimination of high affinity self-reactive T cells (d). Expansion of thymocytes with intermediate affinity for self, into T-regulatory cells (Treg) occurs during interaction of SP thymocytes with DC modified by the cytokine thymic stromal lymphopoietin (TSLP) generated by the Hassall’s corpuscles (HCs) (e). Thymocytes are destroyed during negative selection with DC (f) or survive the conditioning to emerge as recent thymic emigrants (RTE) (g).
Source: Evans VA, Cameron PU, Lewin SR. Human thymic dendritic cells: Regulators of T cell development in health and HIV-1 infection. Clin Immunol 2008;1126:1-12. Used with permission.
Antigen presentation and recognition
Antigen-presenting cells (see below) process foreign proteins into smaller peptides and express these antigens on their cell surface in association with major histocompatibility complex (MHC) molecules, also referred to as human leukocyte antigens (HLA). T cells express either CD4 or CD8 receptors on their surface. These molecules are crucial in antigen recognition and in the function of the T-cell receptor (TcR). The primary function of MHC molecules is the presentation of antigen in the form of short peptides to T cells.
These molecules are essential for T-cell recognition of antigens. MHC-I (HLA-A, B and C) molecules are found on the surface of all nucleated cells and present antigen to CD8+ T cells. MHC-II molecules (HLA-DR, DP, DQ) are expressed only on professional antigen presenting cells such as dendritic cells (DC), B lymphocytes and macrophages. They present antigen to CD4+ T cells. Both MHC-I and MHC-II genes are highly polymorphic.
When the TcR associated with CD3 and CD4 or CD8 molecules, binds to the antigen-MHC complex on an antigen presenting cell, cellular activation signals are transmitted from the complex of TcR/CD3. These TcR activation signals must be accompanied by co-stimulatory signals via CD28 and other co-stimulatory molecules or by cytokines. T-cell receptor signalling alone results in anergy or apoptosis. Activation of antigen presenting cells leads to expression of the classical costimulator molecules including CD40, CD80, CD83 and CD86. Cytokines within the inﬂammatory milieu and signalling from toll-like receptors (TLR) that recognise foreign pathogen associated molecular proﬁles activate the antigen presenting cell to enable effective co-stimulation of T cells.
Note: Depletion of CD4+ T cells in HIV infection may occur as a result of direct infection (*) and death of CD4+ T cells or by indirect mechanisms. HIV-1 infection has been associated with impaired production of CD34 progenitor cells in the bone marrow (a), reduced proliferation of thymocytes and direct infection of CD4+ thymocytes leading to reduced numbers of recent thymic emigrants and CD4+ naïve T cells. In addition, HIV-1 directly infects circulating CD4+ memory T cells although at low frequency (b). At the same time there is significant depletion of mucosal CD4+ T cells (by direct infection of both CCR5+ and CCR5- CD4+ T cells), DCs and macrophages (MΦ) (c). This may potentially compromise the integrity of the mucosal barrier leading to increased translocation of bacteria from the intestinal lumen (d). High levels of immune activation (e) increase the proliferation and death of both CD4+ and CD8+ T cells which are associated with lymph node (LN) fibrosis (f ) and retention of T cells in the LN (g). DN=double negative; DP=double positive; SP=single positive thymocytes.
Source: Evans VA, Cameron PU, Lewin SR. Human thymic dendritic cells: Regulators of T cell development in health and HIV-1 infection. Clin Immunol 2008;1126:1-12. Used with permission.
T-cell effector responses
Upon recognition of cognate antigens binding to the TcR expressed on the cell surface, T cells become activated and develop into effector T cells. CD4+ effector T cells function as helper cells. This help is mediated by the secretion of cytokines which inﬂuence the function of surrounding cells. CD4+ T cells can be broadly divided into subpopulations defined by the pattern of secreted cytokines and surface expression of chemokine receptors that controls their migration to lymphoid and other tissues. CD4+ T cells, in addition to broad classification as helper, cytotoxic or regulatory cells can be described by their stage of maturation as naïve (including recent thymic emigrants), effector memory, central memory, transitional memory, terminally differentiated or stem cell memory. These stages of maturation differ in their longevity and in their patterns of migration, which are largely defined by their surface markers including particularly chemokine receptors and β integrins.
With cognate interactions, the naïve T cells initially develop into non-polarised Th0 memory cells, and with subsequent stimulation develop into type 1 helper (Th1), type 2 helper (Th2) and type 17 helper (Th17) T cells depending on cytokines present in the cellular microenvironment in which they develop.Th1 lymphocytes secrete interleukin (IL)-2 and interferon-gamma (IFN gamma), but not IL- 4, -5 or -6 whereas Th2 lymphocytes secrete IL-4, -5, -6 and -10, but not IL-2 or IFN gamma (Figure 3). Th1 cytokines support cell-mediated immune responses whereas Th2 cytokines generally support humoral immune responses. CD4+ T cells may also exhibit regulatory T-cell (Treg) function and develop in the thymus (central Tregs) or within the peripheral lymphoid tissue. Each of these populations has specific nuclear factors controlling their development (Figure 4).
Note: T cells characteristically possess T-cell receptors (TcR) that recognise processed antigen presented by major histocompatibility complex (MHC) molecules, as shown on the left side of the figure. Most cytotoxic T cells are positive for CD8 receptor, recognise processed intracellular antigen presented by MHC class I molecules, and kill infected cells, thereby preventing or restricting viral replication. Activated cytotoxic T cells secrete interferon-gamma that, together with interferon-alpha and interferon-beta produced by the infected cells themselves, sets up a state of cellular resistance to viral infection. As shown on the right side of the figure, helper T cells are generally positive for the CD4 receptor, recognise processed extracellular antigen presented by MHC class II molecules on antigen presenting cells, and can be divided into two major populations. Type 1 helper (Th1) T cells secrete interferon-gamma and interleukin-2, which activate macrophages and cytotoxic T cells to kill intracellular organisms. Type 2 helper (Th2) T cells secrete IL-4, -5, and -6, which help B cells secrete protective antibodies. B cells recognise antigen either directly or in the form of immune complexes on follicular dendritic cells in germinal centres.
Source: Delves PJ, Roitt IM. The immune system. Second of two parts. N Engl J Med 2000;343:108-17. All rights reserved. Used with permission.
CD8+ effector T cells, on the other hand, are predominantly cytotoxic T cells (CTLs). After binding to antigen on the surface of an infected cell, CTLs kill infected cells by inducing apoptosis by one of two means. Firstly, CTLs may insert perforins into the membrane of an infected cell, facilitating the passage of granzymes which promote apoptosis by direct activation of caspase pathways. Alternatively, CTLs may bind to Fas (a protein which is expressed on the surface of the infected cell) via the Fas ligand (which is expressed on the surface of the CTL). This binding subsequently promotes apoptosis of the infected cell. CD4+ T cells also have the capacity to be cytotoxic under the regulation of CD8+ T cells. Conversely, CD8+ T cells can limit viral replication by non-cytotoxic means, such as the secretion of CD8 Antiviral Factor (CAF), an uncharacterised factor, initially thought to be a defensin.
Note: Thymus-derived naïve CD4+ T cells differentiate into different subpopulations of CD4+ T cells depending on the anatomical site and the milieu in which they encounter antigen presented by dendritic cells (DC). They differentiate into effector or memory cells, with specific nuclear factors required for development of each subset. The mediators produced, and effector functions used, by the cell differ between each subpopulation.
Source: Swain SL, McKinstry KK, Strutt TM. Expanding roles for CD4+ T cells in immunity to viruses. Nat Rev Immunol 2012;12:136-48. Used with permission.
T cell memory
An essential feature of an adaptive immune response is the establishment of immunological memory. Naïve T cells either become effector or memory T cells following their encounter with cognate antigen and DC. This occurs in the lymph node where naïve T cells entering the lymph node from the blood through high endothelial venules (HEV) encounter their cognate antigen carried from the tissue as processed antigens on antigen presenting cells. Following clonal expansion and conversion of naïve T cells to memory T cells, specific changes in the T cell phenotype ensure that the T cells recirculate into peripheral tissues and have a superior quantitative and qualitative immune response upon subsequent encounter with antigen. Memory and naïve cells can be distinguished by cell surface markers. They express different isoforms of the CD45 molecule; CD45RA on naïve cells and CD45RO on memory cells, although naïve cells are more accurately defined by multiple surface markers (CCR7+,CD28+ and CD62L+). Effector memory cells re-express the CD45RA isoform, but can be distinguished from naïve cells in that effector cells do not express CD62L or CCR7. Within the memory CD4+ T cell populations there are subpopulations recognised by the surface expression of specific molecules including CD27 and chemokine receptor CCR7 which is critical in migration of T cells into the lymph node and peripheral tissue. Cells expressing the chemokine receptor CCR6 migrate into gut and peripheral tissues whereas CXCR3 is critical for the migration and T-cell killing at sites of inflammation and virus infection.
Lymph node architecture
The lymph node is a specialised lymphoid organ that facilitates the development of both humoral and cellular adaptive immune responses by providing a site for interaction between recirculating naïve T and B cells and antigen carrying DC migrating from tissue via afferent lymphatics. It is divided into an outer cortex and an inner medulla. The cortex contains B cell-enriched follicles that contain specialised foci called germinal centres. The germinal centres are the site of expansion of antigen-specific B cells and are involved in the generation of antibody responses, in particular the development of B cells with high affinity antigen receptors (immunoglobulin molecules expressed on the cell surface) by a process of affinity maturation and somatic hypermutation of immunoglobulin genes. Follicles contain antigen-presenting germinal centre DC, follicular B lymphocytes, follicular helper CD4+ (TFH) cells as well as a non-haematopoetic population of follicular DC that provide a repository of immune complexes including HIV-containing immune complexes. The T-cell enriched cortical regions between the follicles are known as the interfollicular regions. These thymus-dependent interfollicular regions are enriched in naïve T cells and are critical for the development of cellular immune responses and are the site of initial activation of the naïve T cells during DC-T cell interactions. The T-cell enriched regions contain antigen-presenting interdigitating DC derived from blood or by afferent lymphatics from tissue, CD4+ and CD8+ T cells and some naïve B cells. The collagen matrix and fibroblastic reticular cells provide a scaffolding of the lymph node but in HIV infection there is progressive lymph node fibrosis which is a critical factor in CD4+ T cell loss (Figure 5).
Note: Antigen migrates to the lymph node from infected tissues via the afferent lymphatics as free antigen or processed in tissue and carried by dendritic cells. Naive T cells enter from blood and interact with DC in the cortical T cell zones. B cells interact with T cells and dendritic in the T cell regions and then migrate into the B cell follicle where they expand to produce antigen-specific B cells and plasma cells, which produce antibody. Follicular dendritic cells in the germinal centres of the B cell follicle provide a reservoir of antigen antibody immune complexes. T cells, B cells and plasma cells migrate from the lymph node via the medulla and the efferent lymphatic vessels. Antigen-specific memory or effector B or T cells, activated in the lymph node, can then migrate to tissue sites of infection or inflammation. The collagen matrix and fibroblastic reticular cells network provide the scaffold of the lymphoid tissue.
Source: Estes JD. Pathobiology of HIV/SIV-associated changes in secondary lymphoid tissues. Immunol Rev 2013;254:65–77. Used with permission.
Chemokines derive their name from being chemotactic cytokines. Their principal biological function is to regulate the trafficking of cells to sites of inﬂammation rich in foreign antigens. Broadly, the chemokine family is divided into four subgroups: CC, CXC, C and CX3C where C represents two terminal cysteine residues and X represents the number of intervening amino acids between the two terminal cysteine residues. This structural classiﬁcation also has biological signiﬁcance where CXC chemokines, also referred to as alpha-chemokines, are inflammatory chemokines that attract neutrophils whereas CC chemokines, also referred to as beta-chemokines, are homeostatic chemokines principally involved in homeostatic recirculation and in attracting lymphocytes and monocytes into immune responses.
Chemokines bind speciﬁc chemokine receptors. Most of the chemokine receptors belong to the seven-transmembrane-spanning, G-protein-linked rhodopsin receptor family. Chemokine receptors primarily bind to chemokines within the one class of chemokines and are classiﬁed as being either CC chemokine receptors (CCR) or CXC chemokine receptors (CXCR). Chemokine/chemokine receptor pairs of particular signiﬁcance in HIV pathogenesis are: macrophage inﬂammatory protein-1 alpha (MIP-1 alpha, CCL2), MIP-1beta (CCL3) and ‘regulated on activation, normal T cell expressed and secreted’ (RANTES, CCL5), which are natural ligands for CCR5, and stromal derived factor-1 (SDF- 1, CXCL12), which is the natural ligand for CXCR4. Polymorphisms in these receptor/ligands have been demonstrated to inﬂuence HIV transmission and disease progression (see Natural history of HIV infection).
Immunological aspects of HIV pathophysiology
Most people with HIV infection initially develop HIV-speciﬁc immune responses comprising HIV-speciﬁc CD8+ T cell, CD4+ T cell and B cell activity. These responses are suboptimal and ultimately fail in the majority of people, primarily because of viral immune evasion mechanisms. The preservation of strong humoral and cellular HIV-specific immune responses in those with non-progressive infection does come at the expense of immune activation (see below Long-term non-progression and elite controllers). This does suggest that strategies to restore and maintain host immune responses will be important in the long-term management of patients with HIV disease and for strategies to cure HIV infection. Effective HIV-specific immune responses will also be necessary for the success of prophylactic and therapeutic vaccines (Figure 6).
Note: At the cellular level, CD8+ cytotoxic T cells (CTLs), with the help of CD4+ helper T cells, lyse HIV-infected cells. Soluble factors and neutralising antibodies inhibit viral replication. Macrophage-tropic HIV (R5) infects cells bearing CD4 and the CCR5 coreceptor. T-tropic HIV (X4) infects cells bearing CD4 and the CXCR4 coreceptor. Coreceptors and their ligands (including regulated on activation, normal T cell expressed and secreted (RANTES), the macrophage inflammatory proteins (MIP)-1alpha and MIP-1beta, and stromal cell-derived factor (SDF)-1) are discussed above in section The cellular immune system. At the local level, mucosal CTLs and mucosal immunoglobulin A may inhibit initial viral replication. Concomitant infections activate T cells, providing target cells for HIV to infect. Systemically, there are numerous factors that inhibit HIV replication, including CTLs, antibodies, interferon, and interleukin (IL)-16 and numerous factors that stimulate HIV replication including tumour necrosis factor (TNF), IL-1, IL-6, and co-infections. IL-10 inhibits IL-6 and TNF-alpha (see discussion above in section The cellular immune system). The rectangular bars indicate sites of inhibition. STD = sexually transmitted disease. Source: Hogan CM, Hammer SM. Host determinants in HIV infection and disease. Part 1: cellular and humoral immune responses. Ann Intern Med 2001;134:761-76. Used with permission.
HIV-specific CD8+ T cell responses
CD8+ T cells recognise a variety of peptides from all HIV proteins including reverse transcriptase, envelope, core (gag) and the accessory proteins Vif and Nef, although CD8+ T responses are predominantly directed against epitopes encoded by the gag and env genes. CD8+ T cells recognise divergent HIV strains with greater frequency than HIV-speciﬁc CD4+ T cell responses. The most direct evidence of the critical role of CD8+ T cell responses in the control of HIV and simian immunodeﬁciency virus (SIV) is derived from animal models of macaques infected with SIV. In this animal model, depletion of CD8+ T cells results in rapid rebound of SIV viraemia.
Measurement of CD8+ T cell responses
CD8+ T cell responses can be measured by a variety of assays (Section Immunological diagnostics and therapeutic drug monitoring). Traditional chromium-release assays were used to detect antigen-speciﬁc CD8+ T cell killing of labelled targets. These assays require cell culture and are labour intensive. Other assays to detect antigen-speciﬁc T cells use ﬂow cytometry and granule release assays, tetramers of MHC-peptide, or intracellular cytokine production. Cytokine production can also be detected via enzyme-linked immunospot assay (ELISPOT). Potential discrepancies between the numbers of antigen-speciﬁc CD8+ T cells detected by tetramer staining and those detected by functional assays, such as chromium-release or intracellular cytokine staining, should be appreciated. Chromium-release assays require multiple ex vivo cell divisions and then detection by functional analysis. This may underestimate actual numbers of antigen-speciﬁc CD8+ T cells as these cells may readily apoptose ex vivo. Tetramer assays, on the other hand, may over-estimate CD8+ T cell responses by detecting cells with a TcR binding a specific antigen-MHC complex, which exhibit low cytokine production or cytolytic activity.
Role of CD8+ T cells in HIV pathogenesis
CD8+ T cells play an important role in delaying HIV disease progression. HIV disease progression is associated with a decline in HIV-speciﬁc CD8+ T-cell activity in some patients. Slow progression of HIV disease is associated with strong CD8+ T-cell activity. Furthermore, an inverse correlation between CD8+ T responses and plasma viral loads suggests a protective role for CD8+ T cells. More direct evidence of a protective role of CD8+ T-cell responses in HIV infection comes from animal models. Monkeys subjected to CD8+ T-cell depletion cannot control plasma viraemia during primary SIV infection. Furthermore, CD8+ T cell depletion during chronic SIV infection leads to profound increases in plasma viraemia. These data convincingly demonstrate that CD8+ T cells suppress HIV replication in vivo.
Human leukocyte antigen associations
The association between human leucocyte antigen (HLA) genotype and HIV disease progression underscores the importance of CD8+ T-cell responses in disease pathogenesis. HIV-speciﬁc CD8+ T cells recognise viral peptides when they are presented on the surface of virally-infected cells in association with HLA class I molecules. Different HLA molecules present different peptides. This variation thus inﬂuences the quality of the immune response. For example, HLA-B27 and HLA-B57 are associated with slow HIV disease progression.
A single amino acid change within HLA-B35 is associated with differences in HIV disease progression. Studies on the evolution of viral reverse transcriptase and HLA types have demonstrated that HIV will adapt to HLA type at a population level. Homozygosity of MHC-I antigen genes produces a more restricted CD8+ T cell response than heterozygosity, and has been associated with faster HIV disease progression. An increase in viral load has been associated with mutations in immunodominant epitopes of peptides eliciting CD8+ T-cell responses, commonly referred to as immune escape. This provides further evidence that antigen recognition by HLA is important for viral control.
Recent work has suggested that the mechanism by which HLA molecules inﬂuence disease outcome differs at different stages of the disease. For example HLA-B57 is associated with an early effect in reducing CD4+ T-cell decline and has a persistent effect throughout the disease course. HLA-B27 has no beneﬁt in the early decline in CD4+ T cell counts to < 200 cells/μL but is important later in delaying disease progression. People with HLA-B35 have an accelerated loss of CD4+ T cells early in the disease course but CD4+ T-cell loss is not different to that seen in people with other HLA genotypes at later stages of disease. At least some of these effects are related to the generation of CTL, early suppression of viral replication and the ﬁtness of escape mutants.
Temporal change in CD8+ T-cell responses during HIV progression.
In the majority of people, HIV-speciﬁc CD8+ T cell responses are generated soon after the peak of viral replication during primary HIV infection. Indeed the initial decrease in HIV viral load during the primary illness is attributed to the generation of CD8+ T-cell responses. Detection of HIV-speciﬁc CD8+ T cell responses precedes the detection of neutralising antibodies.
Up to 10% of CD8+ T cells present during primary HIV infection are HIV speciﬁc. However, in other viral infections, such as cytomegalovirus (CMV) and Epstein Barr virus, the proportion of virus-speciﬁc CD8+ T cells can be greater than 10%. The breadth of HIV-speciﬁc CD8+ T-cell responses in acute infection inﬂuences disease progression. Monoclonal expansion of HIV-speciﬁc CD8+ T cells in patients with acute HIV is associated with a poor prognosis. Moreover, once the viral set point is reached following primary infection, there is an inverse relationship between the breadth of HIV-speciﬁc CD8+ T-cell responses and viral load.
HIV-speciﬁc CD8+ T-cell responses are detected in chronic phases of HIV disease in most patients with a frequency of up to 2% of total CD8+ T cells. There are similar proportions of HIV-speciﬁc CD8+ T cells in the lymph nodes. Antigen-speciﬁc CD8+ T cells have two fates. Some cells become terminally differentiated effector cells. Effector cells occur in large numbers, but are not long-lived and die by apoptosis. In contrast, other CD8+ T cells become long-term memory cells. CD8+ memory T cells are in low numbers, but are likely to persist. There may be a continuum of T cells between these two extremes. The CD8+ T-cell effector pool is continuously replenished from the CD8+ long-term memory T-cell pool. In turn, the CD8+ long-term memory T-cell pool is replenished from continued antigen stimulation of the naïve CD8+ T-cell pool. The expansion of CD8+ T cells is in large part dependent on continued antigen stimulation. This may explain why detectable CD8+ T cells decline in parallel with viral load reduction following combination antiretroviral therapy (cART).
HIV-specific CD8+ T-cell functional abnormality
HIV-speciﬁc CD8+ T cells demonstrate a number of functional defects. They are defective in perforin, which is associated with poor ex vivo killing of targets when compared with CMV-speciﬁc CD8+ T cells in the same people. Low levels of perforin have also been observed in CD8+ T cells from lymph nodes in people with HIV disease. It is uncertain why HIV-speciﬁc CD8+ T cells lack or show low perforin expression. One explanation suggests that HIV-speciﬁc CD8+ T-cell differentiation is blocked as these cells lack CD28, but retain expression of CD27. This contrasts with CMV-speciﬁc CD8+ T cells, which lose expression of both markers. The phenotype which lacks CD27 and CD28 expression is thought to indicate mature effector CD8+ T cells. This concept is supported by the demonstration that most HIV-speciﬁc CD8+ T cells are CCR7-CD45RA- (pre-terminally differentiated) compared with CMV-speciﬁc CD8+ T cells which are CCR7-CD45RA+ (terminally differentiated). Failure of CD8+ T cells to mature could be the consequence of impaired CD4+ T-cell help. Disease progression after immune escape in HLA-B*5701-restricted epitopes is associated with the development of reduced perforin production and loss of polyfunctional T cells.
HIV proteins speciﬁcally impair CD8+ T cell responses. Firstly, Nef and Tat decrease HLA class I expression on infected cells. Secondly, Nef increases Fas ligand (FasL) expression on virally infected cells. Interactions between FasL and Fas lead to apoptosis of CD8+ T cells. Finally, Nef inhibits apoptosis in the infected cell by blocking intracellular signalling events downstream of Fas.
Non-cytotoxic CD8+ T-cell responses
CD8+ T cells from people with HIV infection can inhibit the replication of HIV in tissue culture via a non-cytolytic mechanism. This activity has been attributed to the secretion of a cell-associated factor (CAF). The inhibition of HIV replication by CAF has the following features: CAF inhibits HIV replication by non-lytic mechanisms and does not impair the function of the infected cell; it does not alter the activation status of the CD4+ T cell or cause CD4+ T cell proliferation; it acts independently of MHC restriction; it is active against HIV-1, HIV-2 and SIV; and it is active against many different strains of HIV, including both syncytium- and non-syncytium-inducing isolates. CAF inhibits viral transcription by binding to HIV long-terminal repeat (LTR) of viral DNA. In this way CAF interrupts the ability of HIV Tat and host cellular factors to accelerate HIV transcription via interactions with viral LTR.
CAF activity has been demonstrated to correlate with clinical disease progression. People with early HIV disease or non-progressive HIV disease have high levels of CAF. In contrast, people with advanced HIV disease have low levels of CAF. Furthermore, limited longitudinal studies have demonstrated that the loss of CAF is associated with disease progression and that CAF decreases in people on antiretroviral therapy. The alpha-defensins exhibit some CAF characteristics and may account for some of the CAF activity that is not attributable to beta-chemokines15 however the exact mechanism of the anti-HIV effects of alpha-defensins remains unknown. Alpha-defensins and beta-chemokines are produced by cells other than CD8+ T cells, which was not a characteristic of the original description of CAF. The identity of CAF remains a mystery although a number of putative genes have been shown to be differentially expressed in CD8+ T cells showing high CAF activity.
HIV-specific CD4+ T-cell responses
HIV-speciﬁc CD4+ T-cell responses are generated during acute infection in the vast majority of patients, but are markedly reduced or lost in most patients in the chronic stages of HIV disease. Suboptimal CD4+ T-cell help is central to the immunodeﬁciency of HIV disease. Preferential infection of HIV-speciﬁc cells may offer one explanation for poor HIV-speciﬁc CD4+ T cell responses. Similar loss of antigen-specific T cells by infection is seen with vaccination including with the vaccine vector Ad5. The mechanisms leading to the depletion of CD4+ T-cell responses will be critical to the success of attempts to immunologically control HIV disease progression.
Measurement of CD4+ T-cell responses
A variety of in vitro assays are available to measure CD4+ T-cell responses. Traditionally, CD4+ T-cell responses were measured using lymphocyte proliferation assays. In these assays, CD4+ T cells are mixed with HIV antigen and 3H-thymidine. CD4+ T cells which recognise HIV antigens proliferate and incorporate 3H-thymidine. The 3H-thymidine incorporation gives a qualitative measure of CD4+ T cell responses.
Alternatively, CD4+ T-cell responses can be measured by the detection of cytokine production following stimulation with HIV antigens. This may be measured either by ELISPOT assay or by intracellular staining and ﬂow cytometry. These assays give qualitative and quantitative results. In contrast to CD8+ T-cell responses, measurement of CD4+ T-cell responses by tetramer assays has limited availability because of lower affinity of peptide binding to MHC II compared to class I.
Role of CD4+ T-cell depletion and dysfunction in HIV pathogenesis
Loss of effective CD4+ T-cell responses has detrimental consequences for both CD8+ T cells and HIV-speciﬁc B-cell responses. CD4+ T-cell help is crucial for priming CD8+ T cells, maintaining CD8+ T cell memory, and maturing CD8+ T cell function 24 (Figure 7). CD4+ T cells provide this help through the production of local cytokines, such as IL-2 and IL-15 which stimulate CD8+ T-cell activity. CD4+ T cells enhance co-stimulatory pathways between antigen-presenting cells and cytotoxic CD8+ T cells via upregulation of CD40L expression. CD40L is expressed on activated CD4+ T cells. CD40L is crucial in triggering DC to produce IL-12 which, in turn, is central to the initiation of CD8+ T-cell responses. Some viruses can by-pass this step by activation of the DC directly but HIV reduces innate signalling including interferon signalling in myeloid DC. Activation of CD8+ T cells by DC then depends on providing DC-T cell signalling by plasmacytoid DCs that can be directly activated by HIV, produce high levels of IFNα and other type I interferon, that can then activate the myeloid DC and initiate CD8 responses.
No deﬁnite link between lack of CD4+ T-cell help and declining CD8+ T-cell activity has been established in HIV infection, but other models of immunological control of chronic viral infection, such as the murine lymphocytic choriomeningitis model and human CMV disease following bone marrow transplantation, support this theory. However, the observation that viraemia rebounds following withdrawal of antiretroviral therapy, even in patients who preserve HIV-speciﬁc CD4+ T-cell responses following antiretroviral therapy during acute HIV infection, suggests that preservation of HIV-speciﬁc CD4+ T-cell responses is not enough to maintain virological control.
Note: CD4+ T cells are important for priming dendritic cells to initiate CD8+ T cell responses. They help maintain memory cells and are important in maturation of CD8+ T-cell function. All of these actions are impaired by HIV infection. In addition, HIV can directly impair dendritic cell function. Check Fas= function; MIP= macrophage inflammatory proteins; RANTES= regulated on activation normal T cell expressed and secreted; TNF= tumour necrosis factor.
Source: McMichael AJ, Rowland-Jones SL. Cellular Immune responses to HIV. Nature 2001;410:980-7. Used with permission.
Temporal change in CD4+ T-cell responses during HIV progression
HIV-speciﬁc CD4+ T-cell responses are generated early following infection with HIV and are detectable in people treated early in acute infection. In contrast, HIV-speciﬁc CD4+ T-cell responses are low or absent in most people with chronic HIV disease, including those on effective antiretroviral therapy, but are detectable in people with long-term non-progressive disease. HIV-speciﬁc CD4+ T-cell responses have been detected in most people with established HIV disease. However, people have fewer HIV-speciﬁc than CMV-speciﬁc CD4+ T cells, suggesting a limited HIV-speciﬁc CD4+ T-cell response. The decline in HIV-speciﬁc CD4+ T-cell responses is a fundamental process in HIV disease progression.
Several factors are thought to contribute to the impairment of HIV-speciﬁc CD4+ T-cell responses in progressive HIV disease. Possible mechanisms include virus-induced anergy, antigen-induced cell death, direct infection or apoptosis. HIV Tat downregulates HLA class II expression and therefore may lead to viral induced anergy by impairing antigen recognition. An inverse correlation between HIV-speciﬁc CD4+ T-cell responses and plasma viral load suggests that HIV-speciﬁc CD4+ T-cell responses are protective. However, a causal relationship has not been proven. HIV-speciﬁc CD4+ T-cell responses decrease following control of HIV replication with effective antiretroviral therapy, possibly because of reduced antigen presentation. There is abnormal production of T-cell subsets from naïve T cells during HIV infection. HIV-speciﬁc CD4+ T-cell responses are preserved, however, in people who commence antiviral therapy at low viral loads.
CD4+ T cell defects in HIV disease
Progressive HIV disease is characterised by quantitative and qualitative defects in CD4+ T cells manifesting as a decline in both CD4+ T-cell numbers and function. The proximal cause of CD4+ T-cell loss in HIV disease is a perturbation of CD4+ T-cell homeostasis. This perturbation results from both an increase in the destruction of mature effector memory CD4+ T cells and a reduction in the replacement of these cells with precursor cells including central memory CD4+ T cells and immature progenitors. Other factors contributing to the loss of CD4+ T cells in people with HIV include generalised immune activation leading to enhanced T-cell proliferation and death, direct cytopathic effects of HIV and altered trafficking with redistribution of CD4+ T cells to lymphoid organs. A number of factors contribute to the impaired function of CD4+ T cells in people with HIV disease. These include reductions in the proportion of naïve CD4+ T cells, restriction of T-cell receptor repertoire and HIV-induced anergy.
Quantitative defects in CD4+ T cells
CD4+ T-cell homeostasis refers to the process of balance between cell production and destruction resulting in maintenance of stable CD4+ T-cell numbers. Processes which result in new CD4+ T-cell production are referred to as entry pathways and processes which result in the destruction of CD4+ T cells are referred to as exit pathways. These processes are usually tightly regulated, but perturbation of this process leads to CD4+ T cell decreases in people with HIV disease (Figure 2). Predominant entry pathways include CD4+ T-cell production by proliferation of peripheral CD4+ T cells or by differentiation from progenitors via thymic (or extrathymic) pathways. The predominant exit pathways include enhanced proliferation and cell death and HIV-mediated CD4+ T-cell destruction. Over time in untreated people, exit pathways predominate, resulting in a median CD4+ T-cell decline at a rate of 84 cells/μL per year.
Alterations to entry pathways
Increased CD4+ T cell proliferation
Untreated HIV infection is characterised by marked increases in immune activation and lymphocyte proliferation which exceed that observed in controls without HIV infection by at least two- to three-fold. This augmented proliferation is predominantly observed in memory CD4+ (and CD8+) T cells, which proliferate rapidly in comparison to naïve T cells. Although recent work suggests that gut derived microbial products drive proliferation (see Immune activation below) other causes of peripheral lymphocyte proliferation include: HIV-antigen speciﬁc expansion; stimulation of T cells by cross-reactive antigens; and cytokine-mediated bystander activation. Proliferating cells are susceptible to activation-induced apoptosis which leads to eventual decline in CD4+ T-cell numbers, if not compensated for by other entry pathways (see below). Increased lymphocyte proliferation induces anergy (see below). Thus, increased proliferation of CD4+ T cells has negative effects in people with HIV.
Reduced CD4+ T cell differentiation from progenitor cells
Contrary to previously held beliefs that the thymus is not active in adults, many reports demonstrate that substantial thymic output persists even into late adulthood. However, the role of the thymus in adults with HIV infection is poorly understood. Thymic function may be measured by imaging (e.g. CT scan), histology and the measurement of T-cell receptor excision circles (TREC, i.e. episomal circular DNA products produced following T-cell receptor gene rearrangement during thymic development) in blood T cells. When peripheral CD4+ T-cell proliferation is taken into account, these measures of thymic function suggest that thymic function is reduced in people with HIV disease. While thymic size is decreased in people with advanced HIV disease, people with moderate HIV-related immune deﬁciency have more thymic tissue than age-matched controls without HIV infection. Furthermore, the amount of thymic tissue correlates with the rate of increase in naïve CD4+ T cells following antiretroviral therapy. These ﬁndings suggest that increased production of naïve CD4+ T cells by the thymus in people with early HIV disease initially compensates for the loss of CD4+ T cells secondary to increased peripheral turnover. However, thymic function wanes over time (perhaps because of viral factors discussed below), which leads to a net decrease in the size of the CD4+ T-cell pool.
Alterations to exit pathways
HIV-induced CD4+ T-cell loss in blood and tissues
HIV induces cell death in both infected and uninfected bystander CD4+ T cells by cytopathic, apoptotic and immune processes. Mechanisms underlying HIV-induced CD4+ T-cell loss are outlined in Table 1. Despite a marked increase in CD4+ T-cell turnover in HIV disease, direct HIV cytopathic effect on CD4+ T cells in blood has been difficult to demonstrate.
Studies of gastrointestinal tract lymphoid cells however have shown a rapid and profound loss of CD4+ T cells in acute HIV infection in humans and SIV infection in primates. CD4+ memory T cells are lost primarily in lamina propria of the gut mucosa. This happens as early as days 4 to 14 of acute infection. Less than 20% of the cells are lost by direct infection or by cytotoxic lymphocyte activity. The predominant mechanism of cell loss is a bystander effect by gp120 induced FasL-mediated apoptosis and is speciﬁc for CD4+ T cells. CD4+ T-cell depletion in the gut has been demonstrated at all stages of HIV disease and accounts for a signiﬁcant and sustained loss of total body CD4+ T cells. The gut depletion does not occur in long term non-progressors who maintain CD4+ T cells and suppress viral replication The effect of antiretroviral therapy on depletion of CD4+ memory T cells in the gastrointestinal tract is variable, although reconstitution is more effective following treatment in primary infection, and reconstitution of gut Th17 cells may also occur with therapy.
Redistribution of CD4+ T cells
Alterations to physiological pathways of lymphocyte trafficking may account for some changes observed in the peripheral blood of people with HIV. CD4+ T cells are sequestered in lymph nodes of people with untreated HIV infection. Antigenic stimuli within lymph nodes result in the activation and retention of CD4+ T cells within the lymph node. Increased chemokine expression in the lymph nodes of people with untreated HIV disease and ﬁbrosis may contribute to this phenomenon. The initial phase of CD4+ T cell increase following initiation of cART is thought to represent the corresponding redistribution of CD4+ T cells from lymph nodes into the peripheral blood.
Qualitative defects in CD4+ T cell function in HIV disease
Memory and naïve T cells
In addition to the quantitative defects in CD4+ T cells, there are many qualitative defects in progressive HIV disease. There is a decrease in the proportion of naïve CD4+ T cells (CD4+CD45RA+CD62L+) and a corresponding increase in the proportion of memory CD4+ T cells (CD4+CD45RO+). This alteration may be secondary to reduced thymic production of naïve CD4+ T cells and an increased peripheral proliferation of memory CD4+ T cells. Preferential HIV infection of naïve CD4+ T cells with cytopathic HIV variants may also contribute to the relative decrease in naïve CD4+ T-cell numbers.
Second, there is a restriction in the T-cell receptor repertoire, as a result of deletion of some antigen-speciﬁc CD4+ T-cell clones following antigen-induced cell death.
CD4+ T-cell function in people with HIV is also largely limited by anergy, functionally deﬁned as a state of hyporesponsiveness in which lymphocytes do not respond appropriately following antigenic stimulation. Anergy is present at all stages of HIV disease and can be demonstrated before any decline in CD4+ T-cell counts. It is measured in vivo (by cell mediated immunity skin testing) and in vitro (by antigen stimulation assays). Anergy is a state of unresponsiveness that results from a number of integrated signals including those from suboptimal TcR stimulation, reduced co-stimulation, increased TREG signalling and negative regulators or immune check points such as PD-1. Some of the putative processes involved in the development of anergy are depicted in Figure 8. While the mechanisms underlying anergic processes in HIV infection are ill-deﬁned, many are reduced following the initiation of antiretroviral therapy.
In HIV infection, there is altered expression of T-cell molecules that lead to an impaired antigen-speciﬁc response, commonly referred to as T-cell exhaustion. Increased expression of CTLA4 and PD-1 (Programmed Death-1) on both CD4+ and CD8+ T cells contribute to reduced immune responses in people with HIV infection. Blocking the PD-1 pathway allows recovery of CTL responses.
Regulatory T cells
TREG cells are also important in regulating antigen-speciﬁc immune responses. TREG cells are generated in the thymus or in the periphery and are marked by expression of an intracellular marker FoxP3. TREG cell numbers are regulated in the thymus by DC, in particular those developing in the presence of thymic stromal lymphopoetin (TSLP). TREG cells express CD4, CD25 and intracellular FoxP3, have reduced surface expression of CD127, are susceptible to HIV infection and may be depleted from the gut early after infection. Loss of TREG cells may be associated with increased T-cell proliferation although this has not been a consistent ﬁnding. The persistence or expansion of antigen speciﬁc TREG may block antigen speciﬁc CTL.
Humoral immune responses against HIV
All people generate antibody responses against HIV. While the clinical relevance of these antibodies in established HIV infection remains to be defined, the generation of antibodies for prophylactic vaccine strategies is thought to be critical. The production of effective broadly neutralising monoclonal antibodies and the mechanisms for their generation is seen as the pathway to more effective antibodies.
HIV specific antibodies
Currently, HIV infection is diagnosed by detection of HIV antibodies using enzyme-linked immunosorbent assays (ELISAs). Currently used ELISAs allow identiﬁcation of antibodies that bind to HIV antigens and detection of HIV antigens, specifically HIV p24. Antibodies to HIV are characterised further by demonstrating which viral proteins the antibodies bind to using Western blot assays. HIV antibodies detected by ELISA and confirmatory Western blot assay may not have functional activity, as opposed to neutralising antibodies that inactivate or neutralise HIV replication. The appearance of HIV-binding antibodies detected by the ELISA and Western blot assay (see section Virological diagnostics) occurs prior to the appearance of neutralising antibodies. The ﬁrst HIV-speciﬁc antibodies detected are those directed against the structural, or gag, proteins of HIV, p24 and p17, and the gag precursor, p55. The development of antibodies to p24 is associated with a decrease in the serum levels of free p24 antigen. Antibodies to the gag proteins are followed by the appearance of antibodies to the envelope proteins (gp160, gp120, p88 and gp41) and to the products of the pol gene (p31, p51 and p66).
Broadly neutralising antibodies
Broadly neutralising antibodies are those reactive with a broad range of virus isolates. These can be generated in many subjects including some controllers of HIV but they are generally related to high viral load and to chronic antigen exposure and develop later in the course of infection. A number of these antibodies have been generated as monoclonal antibodies and used therapeutically to control or block infection in non-human primates  and humans  These include antibodies to the CD4 binding site, the membrane proximal region binding to gp41, glycan and amino acid residues in the V1, V2 and V3 regions of the HIV env. These monoclonal antibodies require the ability to bind to Fc receptors for optimal efficacy suggesting that they may be functional by mechanisms involving FcR bearing cells and by antibody-dependent cellular cytotoxicity (ADCC). Further it is now clear that broadly neutralising antibodies have the ability to bind to the gp120 trimer compared to non-neutralising gp120 antibodies that bind to monomeric gp120. Recent work has focused on the pathways for the development of such broadly neutralising antibodies. These broadly reactive antibodies characteristically have long complementarity determining regions (CDRs) and are hypermutated indicating that they depend on germinal centre reactions and T follicular helper lymphocytes. Sequential vaccination strategies have been proposed that may lead to the development of such antibodies in vaccinated people.
HIV-speciﬁc neutralising antibodies bind to HIV and inactivate it
Several weeks after the decrease in viraemia following primary infection, neutralising antibodies are detectable, and primarily target proteins expressed on the viral envelope. There are conﬂicting data concerning the correlation of HIV-speciﬁc neutralising antibodies and HIV disease progression. Some studies have demonstrated that people with long-term non-progressive HIV disease have high levels of antibodies that are capable of neutralising a large number of HIV isolates. It has been suggested that a constantly changing viral quasispecies in this patient group may give rise to a wide range of epitopes for antigen presentation. In contrast, people with progressive HIV disease have low or absent antibodies, which neutralise autologous strains.
Neutralising antibodies have not been detected in highly exposed but persistently seronegative (HESN) people. Several small studies have provided some evidence to suggest that HIV-speciﬁc neutralising antibodies may be protective against HIV disease progression. For instance, the development of neutralising antibodies was associated with a reduction in viral load in health-care workers who acquired HIV infection following needlestick injuries. There is disagreement in the literature regarding any protective effect of maternal neutralising antibodies in HIV-exposed infants. Finally, passive administration of neutralising antibodies protected macaques from SIV infection and reduced viral rebound after treatment in humans with HIV infection.
A number of factors limit the development of effective HIV-speciﬁc neutralising antibodies. These include the poor antigenicity of HIV-envelope proteins and the fact that critical sites such as the CD4 receptor and co-receptor binding site are hidden as a result of glycosylation on the complex three dimensional structure of gp120. Broadly neutralising antibodies inhibit viral function in three ways: they induce the dissociation of gp120 from gp41; they directly inhibit viral binding to receptor- coreceptor complexes; and they interfere with post-attachment steps of gp41, which lead to virus-membrane fusion. Human studies have conﬁrmed the importance of the membrane proximal region of env as a target resistant to viral escape.
Antibody-dependent cellular cytotoxicity (ADCC)
Antibodies to both gp120 and gp41 participate in antibody-dependent cellular cytotoxicity (ADCC)-mediated killing of HIV-infected cells. The concentration of anti-envelope antibodies capable of mediating ADCC is highest in the early stages of HIV infection. ADCC-mediated cytotoxicity correlates with control of HIV viraemia during acute infection. The protective effect of neutralising antibodies in acute infection depends on FcR but not complement binding. This is consistent with protection by both activity on free virus and clearance of cells binding antibody by mechanisms involving ADCC.
Other cells and contributors to HIV pathogenesis
Lymph node structure
There are three clearly deﬁned stages of lymph node destruction in HIV disease: follicular hyperplasia; follicular disruption; and follicular depletion. High levels of HIV RNA are present in the lymph nodes at all stages of HIV disease.
In the ﬁrst stage, follicular hyperplasia (type I), the lymph node has the appearance of a normally reactive node. The lymphoid follicles enlarge and coalesce and are associated with markedly hyperplastic germinal centres. HIV RNA is conﬁned within the germinal centres. HIV is attached to follicular DC via antibody in immune complexes associated with complement. HIV-induced follicular expansion is characterised by the predominant expansion of B cells associated with T follicular helper cells which are a target for HIV infection and may be a reservoir for HIV replication. Follicular hyperplasia is generally associated with peripheral blood CD4+ T cell counts > 500 cells/μL.
The next stage, follicular disruption (type II), is characterised by gradual loss of germinal centres, thickening of the lymph node capsule and irregularly distributed proliferation of small blood vessels. Nodal architecture is distorted with a polymorphic population of lymphoid cells, plasma cells and multinucleated giant cells. HIV replication is no longer contained within the germinal centres. Follicular disruption (type II) generally correlates with a CD4+ T cell count of 200-500 cells/μL.
In the third stage, follicular disruption (type III), the germinal centres are completely involuted and the lymph node architecture completely destroyed. There is striking lymphocytic depletion and reduction in lymph-node size. Excessive vascularisation, diffuse ﬁbrosis and plasma-cell inﬁltration characterise the inter-follicular regions. Appropriate T-cell responses are decreased in late-stage HIV disease secondary to the destruction of the stromal microenvironment of lymph nodes. The use of inhibitors of fibrosis have been shown to be effective in reducing T-cell loss in non-human primates  and are in clinical trials in patients.
HIV infection of monocyte-derived macrophages leads to sustained infection with limited cytopathic effect. Monocyte/macrophage infection contributes only 1% to the overall viral load in untreated people. However monocyte/macrophages play a critical role in the pathogenesis of HIV disease largely as a result of impaired function. HIV-infected macrophages are an important reservoir in people on cART.
HIV-infected monocytes/macrophages as well as DC contribute to CD4+ T-cell depletion by multiple means including: gp-120 interactions with the CD4 receptor on neighbouring T cells resulting in cellular fusion; increased FasL expression; and HIV infection of neighbouring CD4+ T cells. HIV infection of macrophages and microglia are important in the pathogenesis of HIV encephalopathy. Furthermore, HIV impairs macrophage-effector functions, thereby contributing to development of opportunistic infections, such as toxoplasmosis  and Candida albicans.
Monocytes/macrophages from people with HIV infection have reduced expression of MHC class II and co-stimulatory molecules, which impair antigen presentation. HIV infection of monocyte/macrophages is also associated with downregulation of macrophage-surface receptors (e.g. mannose receptor) that play an important role in internalisation of pathogens.
Monocyte subpopulations that express low CD14 and high CD16 have the ability to produce higher levels of pro-inﬂammatory cytokines. This population of cells is more susceptible to HIV infection than CD14 high monocytes in vitro and is more frequently infected during HIV infection in vivo. These cells may be the predominant monocyte population that is recruited into the central nervous system (CNS) and contribute to the development of the HIV-associated neurological disease.
Natural Killer cells and gamma-delta T cells.
Natural Killer (NK) cells are activated in the absence of MHC class I and provide a protection against virus infections when CTL responses are ineffective. Loss of MHC class I molecules, such as occurs in HIV-infected cells producing Nef, will result in NK targeting and cytolysis by mechanisms shared with CTL. HLAE is expressed speciﬁcally on infected CD4+ T cells and is recognised by gamma-delta T cells. Gamma-delta T cells play a role in controlling the HIV viral load. NK cell activation is associated with expression of TRAIL, a TNF family molecule that induces apoptotic cell death in TRAIL-ligand expressing target cells. NK cells also have a immunoregulatory effect as they are responsible for the ﬁnal destruction of DC in lymph nodes and thus will limit the expansion of T cells.
HIV infection is associated with abnormal activation of B cells. Multiple viral proteins, in particular gp41, induce polyclonal B cell activation. Hypergammaglobulinemia develops and is particularly evident in children. Polyclonal B cell hyperactivity during the early phases of HIV infection may be the major process predisposing to B cell malignant transformation and AIDS-associated lymphomas.
HIV-speciﬁc CD8+ T cells provide the cornerstone of the host immune response to this virus. HIV-speciﬁc CD8+ T-cell responses are generated early in acute infection and persist into the chronic phases of HIV disease. HIV-speciﬁc CD8+ T-cell responses are directed against a broad range of HIV peptides and have been demonstrated to control SIV viraemia in animal models. The inﬂuence of HLA class I on HIV disease progression underscores the importance of CD8+ T-cell responses in HIV disease pathogenesis. In contrast, HIV-speciﬁc CD4+ T-cell responses, while being generated in the acute stages of HIV disease, are absent or severely impaired in the vast majority of people with chronic HIV disease. Impairment of HIV-speciﬁc CD4+ T-cell responses is one factor that leads to the waning of HIV-speciﬁc CD8+ T cells. The contribution of humoral immune response to HIV is less clear. While HIV-speciﬁc neutralising antibodies are detected in most people, these antibodies rarely neutralise concurrent autologous strains of HIV. Nonetheless, given the capacity of passive administration of broadly neutralising antibodies to protect against infection in animal models and in humans, the generation of HIV-speciﬁc neutralising antibodies is seen as a critical component of successful prophylactic vaccine strategies.
Immunological control in specific patient groups
Highly exposed but persistently seronegative individuals
The observation that some people remain HIV antibody-negative despite repeated exposure to HIV suggests that mechanisms exist to protect humans from HIV infection. Putative immunological and genetic associates of protection from HIV infection have been identiﬁed in cohort studies of infants born to mothers with HIV infection, exposed health-care workers, and sexual partners of people with HIV infection including sex workers in areas of high HIV prevalence. In these studies, HIV infection is excluded by the absence of HIV DNA and the inability to culture virus from peripheral blood mononuclear cells.
Exposure to HIV in a subset of people may actually prevent subsequent infection by stimulating an effective HIV-speciﬁc CD8+ T-cell response. HIV-speciﬁc CTLs have been identiﬁed in 30% of highly exposed but persistently seronegative (HESN) men who have sex with men, heterosexual partners of people with HIV infection, infants of mothers with HIV, health-care workers following needlestick injuries containing HIV-infected blood and sex workers.
While HIV-speciﬁc CTLs persist for up to 34 months following HIV exposure, repeated exposure to HIV is thought to be necessary for the maintenance of HIV-speciﬁc CTLs. Longitudinal studies have demonstrated that HIV-speciﬁc CTLs wane during periods of reduced HIV exposure. HIV seroconversion has been reported in sex workers during periods of reduced HIV exposure and waning HIV-speciﬁc CTLs despite previously strong HIV-speciﬁc CTL.
Interestingly, HESN people recognise CTL epitopes rarely recognised by people with HIV infection. Moreover, cross-clade HIV-speciﬁc CTL recognition of conserved epitopes has been demonstrated in some people. This suggests that vaccine development should include epitopes recognised in these people. HLA restriction has been demonstrated for exposed sex workers without HIV infection. Similarly, babies without HIV infection have demonstrated restricted HLA type and decreased HLA concordance with their mothers with HIV infection.
In addition, exposure of macaques to subinfectious inocula of SIV-induced SIV-speciﬁc T-cell responses was associated with protection of the animals from subsequent infection. Taken together these data suggest that HIV-speciﬁc CTL play important roles in preventing infection.
Non-cytotoxic HIV-speciﬁc immune responses may also play a role in preventing HIV infection in exposed people. HIV-speciﬁc CD4+ T-cell proliferative responses occur in up to 75% of the people in the cohorts described above. Furthermore, increased production of beta-chemokines, which may limit HIV entry into susceptible cells, occurs in highly exposed people, as does non-cytotoxic CD8+ T cell-mediated HIV suppression. The detection of HIV-speciﬁc antibody responses in HESN people is described in some but not all studies. Local factors including serpin, trappin-2 and innate immune responses including low PRR have been found to be present in HESN people and HIV-specific IgA antibodies have been found in vaginal fluid of HESN women participating in a microbicide trial.
Host genetics may have a role in HIV transmission but chemokine receptor polymorphisms account for only a small percentage of exposed people without infection. For example, CCR5 ∆32 homozygosity has been reported in up to 3% of exposed people without HIV infection.
Many studies have examined factors associated with slow progression to advanced HIV disease and have identiﬁed heterogenous groups designated long-term non-progressors (LTNP). These may include slow progression because of defective virus (e.g. deletion of the nef gene) or because of favourable host factors. Individuals who maintain an undetectable viral load in the absence of antiretroviral therapy (approximately 1 in 300 individuals with HIV infection) have been called elite controllers (EC).
Adaptive and innate immune responses appear to be more important than viral factors in elite controllers. The virus replicates efficiently in vitro and sequence analysis shows no demonstrable genome defects. HLA antigens associated with a better prognosis, including HLA-B57 and B27 are found more frequently in elite controllers. Although there is no detectable virus, elite controllers do show a gradual loss of CD4+ T cells. Elite controllers also have a lower level of immune activation and lipopolysaccharide (LPS) than viraemic individuals but levels are higher than that observed in people without HIV. Finally, recent studies have shown that survival of central memory T cells is enhanced in elite controllers compared with people with HIV on cART suggesting that elite controllers have a greater capacity to replenish HIV-infected effector memory T-cells and maintain normal T-cell numbers. The detection of DNA by intracellular sensor cGAS is effective in conventional DC of elite controllers which leads to type I IFN and upregulation of interferon stimulated genes (ISG). This results in effective CD8+ CTL production. Recent work has suggested that the germinal centre and the B cell follicles may be the critical reservoir for persistent productive infection in elite controllers.
A group of patients maintaining low or undetectable HIV viral load following discontinuation of antiretroviral therapy for primary HIV infection has been identified in the VISCONTI study. This group of patients does not have the genetic factors associated with control of HIV replication in elite controllers and has reduced immunological responses to HIV by antibody and T cells. Preservation of central memory CD4+ T cells has been demonstrated in this group of patients. Modelling of the viral load in such patients suggests that this may represent a state where the viral reservoir is reduced below the level required to initiate further infection or to restimulate immune responses. It is unclear how frequent this early control of virus by antiretroviral therapy may be in patients on long-term therapy after early initiation of treatment.
HIV and escape from host immune responses
Multiple mechanisms exist by which HIV evades speciﬁc immune responses. These escape strategies are mutational or constitutive, and result in the evasion of cellular and humoral immune responses.
HIV-specific CTLs kill HIV-infected cells following recognition of viral epitopes presented in association with MHC class I molecules on the surface of virally infected cells. Epitope deletion, secondary to viral mutation, occurs in primary infection and later stages of HIV disease, and results in the loss of antigen recognition by HIV-speciﬁc CTLs (Figure 9). Multiple lines of evidence suggest that mutational escape plays an important role in HIV disease pathogenesis. Viral mutants which have escaped HIV-speciﬁc CTLs are more frequently found in mothers who transmit HIV to their infants than in non-transmitting mothers. Furthermore, following autologous transfusion of HIV-speciﬁc CTL clones directed at a single HIV epitope, viral mutants evolve that lack the targeted epitope and thus escape control of the transfused HIV-speciﬁc CTLs. Viral escape from HIV-speciﬁc CTLs is associated with a poorer clinical outcome. At the population level, escape from CTL determines the distribution of viral sequences so that the predominant sequences are those that have escaped from the predominant HLA alleles in the population. HIV escape from antibody responses has also been demonstrated, but is less well studied than escape from HIV-specific cytotoxic T cells. Emergence of viral mutants which resist neutralisation by autologous HIV-specific antibodies has been demonstrated in people with symptomatic primary infection. Neutralising antibodies have been found in some people with long- term slowly progressive HIV disease, but these antibodies lose their capacity to neutralise HIV over time. Serum fails to neutralise concurrent viral isolates present in people with long-term infection, despite neutralisation of earlier isolates. Broadly neutralising antibodies are now considered to be the result of viral escape from neutralising antibodies. These observation suggest viral isolates continually evolve to resist the humoral immune response.
Note: The propensity of HIV for variation allows it to avoid HIV-specific cytotoxic lymphocyte (CTL) recognition by a number of mechanisms. HIV-specific CTL selective pressure can select for epitope deletion by: 1) changes in the amino acid residues which flank an epitope leading to altered proteolysis and epitope loss; 2) mutations in the major histocompatibility complex (MHC) anchor residues leading to reduced binding to MHC class I molecules and loss of cell-surface presentation; 3) variation in the peptide-MHC class I surface recognised by the T cell receptor (TCR) leading to TCR antagonism, CTL anergy, distortion of the CTL repertoire, and CTL decoy activity.
Source: Sewell AK, Price DA, Oxonius A, Kelleher AD, Phillips RE. Cytotoxic T lymphocyte responses to human immunodeficiency virus: control and escape. Stem Cells 2000;18:230-244. Used with permission.
HIV has developed other means to evade HIV-speciﬁc immune responses. First, post-integration viral latency protects the infected cell from immune surveillance, as viral antigens are not expressed on the cell surface. These latently infected cells produce infectious HIV following subsequent cellular activation. Second, a number of HIV proteins interfere with critical cellular processes that facilitate the host immune response. Speciﬁcally, the HIV tat protein impairs antigen processing by interfering with proteasome function and downregulating MHC class II expression while the HIV Nef protein downregulates CD4 receptor and MHC class I molecule expression. Finally, HIV proteins, speciﬁcally Nef, induce apoptosis of HIV- speciﬁc CTLs by increasing the expression of FasL, resulting in apoptosis of Fas-expressing CTLs. This process is referred to as back-killing (Figure 10).
Note: HIV has a number of constitutive mechanisms to counteract the antiviral effects of cytotoxic lymphocytes (CTLs). These include:
- downregulation of major histocompatibility complex class I;
- destruction of T help for HIV-specific CTLs, interference with antigen processing and upregulation of Fas ligand to ‘back-kill’ antiviral CTLs;
- pre-integration latency; and
- post-integration latency.
Source: Sewell AK, Price DA, Oxonius A, Kelleher AD, Phillips RE. Cytotoxic T lymphocyte responses to human immunodeficiency virus: control and escape. Stem Cells 2000;18:230-244. Used with permission.
Mucosal changes and microbial translocation
Enterocytes of the gut epithelium show evidence of apoptosis very early after infection with a peak 14 days following infection. This apoptosis is accompanied by increased epithelial proliferation and the development of regenerative villous enteropathy, which is characterised by villous atrophy plus micro-abscesses. This process is driven both by T cell activation and failure of mucosal regeneration. The compromised mucosal integrity may potentially lead to an increase in circulating bacterial products as described in graft versus host disease and inﬂammatory bowel disease. Gut permeability is increased 2 to 10 fold in people with HIV infection. Chronic HIV disease is associated with increased levels of LPS, a marker of gram negative microbial products, compared with seronegative people and those with early HIV disease. Plasma levels of LPS detected in people with chronic HIV were also correlated with other markers of systemic immune activation including HLA-DR and CD38 expression on CD8+ T cells and plasma IFNα levels and soluble CD14. LPS binds to monocytes via CD14, which is released as soluble CD14 by activated macrophages. Blood levels of intestinal fatty acid binding protein (I-FABP) also correlates with microbial translocation and inflammation as does the levels of tryptophan metabolites. Microbial products may lead to enhanced immune activation by: increase in cytokine production by antigen presenting cells (IFNα and IL-15); increase in the production of cytokines IL-2, IL-4, IL-7 and IL-15 by lymph node cells; and direct activation of plasmacytoid DC. Direct ligation of TLR by bacterial products activates CD4+ and CD8+ T cells to express CD38, CD69 and to drive CD4+ T cells into cycle. TLR2 ligands have been shown to directly induce susceptibility to HIV infection, particularly by CCR5 using HIV-1. Thus microbial translocation may also increase viral replication and induce T cell loss by indirect mechanisms.
Non-pathogenic and pathogenic non-human primate models
Further data on the role of immune activation in HIV pathogenesis come from African green monkey and sooty mangabey monkey models. Both these animals are infected naturally with SIV, and develop high viral loads but do not lose CD4+ T cells or show development of AIDS. In contrast, SIV infection of rhesus macaques leads to high viral loads, CD4+ T cell depletion and the development of AIDS as found with HIV infection in humans. Levels of immune activation differ but even in those cases where sooty mangabey monkeys have persisting CD4+ T cell depletion there is no clinical disease. Interestingly, in sooty mangabey monkeys there is a rapid depletion of the gut-associated CD4+ T cells in common with pathogenic infection.
Non AIDS disease and deaths
Immune activation may not only lead to CD4+ T cell decline and impaired host immune responses against opportunistic infections but may also lead to serious non-AIDS events. Chronic immune activation may lead to atherosclerotic vascular disease of the heart and brain, osteoporosis and renal disease that have become increasingly apparent since antiretroviral therapy reduced the incidence of most opportunistic infections and AIDS-related malignancies by up to 80%.
Antiretroviral therapy alone, by suppressing viral replication, results in marked but incomplete restoration of cell-mediated immunity. Moreover, HIV-speciﬁc immune responses decline in people taking antiretroviral therapy. In some individuals receiving antiretroviral therapy, restoration of total CD4+ T cells is also impaired despite excellent virological control. Current antiretroviral strategies are limited by toxicity, cost, compliance, resistance and need for continuous therapy. The incomplete recovery of cell-mediated immune function following antiretroviral therapy are related to: persistent immune activation; loss of HIV-speciﬁc immune responses; persistent HIV-induced immunodeﬁciency; and HIV-associated anergy. Immune-based therapies may prove to be useful adjuncts to antiretroviral therapy in augmenting and accelerating improvements in cell-mediated immune function.
The data from the monkey models, including African green monkeys and sooty mangabey monkeys, suggest that evolution of host factors has allowed the persistence of SIV as a non-lethal infection. This adaptation seems to have occurred in the presence of high viral load and results in reduced immune activation and apoptotic loss of T cells. Non-immunological factors control retroviral replication including the APOBEC system and the TRIM5alpha system. These represent innate mechanisms that control retroviral infection including genomic repetitive elements and endogenous retrovirus. A recent whole genome approach showed no association with acquisition of HIV other than the effect of CCR5Δ32. By this same approach, disease progression was related most strongly to the well known HLA-B57 association. The protective change was also linked to an endogenous retroviral element (PC5), an HLA-C related determinant and an RNA polymerase subunit gene.
Boosting HIV-specific immune responses
Strategies to restore and maintain HIV-speciﬁc immune responses may be critical in the long-term control of HIV disease. Controversy persists regarding the essential components of the immune responses needed to prevent disease progression or to clear virus in patients on antiretroviral therapy, the identity of immunogenic epitopes that will stimulate broad immune responses, and the identity of in vitro correlates of immune protection. The success of boosting HIV-speciﬁc immune responses will require the stimulation of both CD4+ T and CD8+ T cell responses; the stimulation of broad responses directed towards multiple epitopes with polyfunctional T cells; and the concurrent use of antiretroviral therapy to limit HIV replication. A recent CMV vector vaccine used in SIV infection of non-human primates looks the most promising of the existing vectors.
Approaches to boosting HIV-speciﬁc host immune responses by use of CTL expanded in vitro have been unsuccessful. However, expansion of CTL and CD4+ T cells in vivo using DC vaccination is clearly effective in reducing viral load. Simpler therapeutic vaccination strategies that use exogenous or endogenous antigens are being developed. In contrast, the hope that intermittent cessation of antiretroviral therapy might potentially regenerate HIV-speciﬁc immune responses has been discarded because of the clear reduction in survival and lack of clear efficacy of this strategy. Similar approaches to induce high level neutralising antibody have some potential application.
Use of immune modulators, including cytokines, to reduce immune activation and improve CD4+ T cell recovery have had only modest effect compared to antiretroviral therapy and does not have a clear role at present (Tables 2 and 3).
|IL-2||Increase T cell proliferation Increase T lymphocyte apoptosis Increase thymic function Increase HIV-specific responses (CAF) Increase antigen presentation|||
|IL-7||Increase thymic-medicated T lymphocyte differentiation and homeostatic T cell proliferation|||
|IL-10||Decrease pro-inflammatory cytokine production|||
|IL-12||Increase HIV and non-HIV CTL responses|||
|IL-15||Increase effector memory CD4+ T cells Increase CTL perforin expression Increase T cell replication and virus production|||
|IL-16a||Increase responsiveness to IL-2 Decreased activation-induced apoptosis|||
|IL-21||Increase CTL perforin expression Decrease TREG production by TGF-beta Increase TH17 cells|||
|GM-CSF||Increase macrophage anti-HIV activity Vaccine adjuvant|||
|KGF||Increase thymic epithelial cell production of IL-7|||
|Growth Hormone||Increase thymic tissue|||
a These cytokines also have direct anti-HIV effects as suggested by inhibition of HIV replication in vitro. CAF = cell-associated factor; CTL = cytotoxic T lymphocyte; IL = interleukin; Th1 = type 1 helper, KGF = keratin growth factor; TGF = transforming growth factor.
|Agent||Mechanism of action and rationale||References|
|Corticosteroids||Decrease pro-inflammatory cytokine production Decrease activation induced lymphocyte death|||
|Cyclosporin||Decrease target cells by decreasing IL-2 production, cellular activation and proliferation Decrease viral maturation by interaction with immunophillins|||
|Hydroxyureaa||Decrease cellular proliferation by decreasing intracellular nucleotide concentration. No clinical benefit.|||
|Mycophenolate mofetil||Decrease lymphocyte proliferation to antigenic stimuli Increase apoptosis of activated lymphocytes|||
|Thalidomidea||Decrease production of TNF-alpha Reduces CCR5 and CXCR4 upregulation by HIV|||
|Rapamycin||Acts through inhibition of mTOR to reduce T cell activation, apoptosis and autophagy|||
|Statins||Atorvastatin reduces immune activation by decreasing T cell signalling. Pravastatin does not have this effect.|||
|Hydroxychloroquine||Hydroxychloroquine and chloroquine act on TLR signalling in pDC to reduces immune activation. Limited or no effects on CD8+ T cell activation or CD4+ T cell recovery in antiretroviral therapy-treated patients and CD4+ T cell depletion in untreated patients.|||
|Cyclooxygenase-2 (COX-2) inhibitors||Reduced LPS-induced upregulation of COX-2|||
a Because of effects in nucleotides, these agents may augment effects of antiretroviral agents. TNF = tumour necrosis factor.
Eradication of HIV cellular reservoirs and cure.
Immune-based therapies were initially trialled as alternatives or adjuvants to antiretroviral therapy, particularly in patients with CD4+ T cell depletion. Subsequently immune modulators have been used to reduce the immune activation associated with HIV infection and in particular gut bacterial translocation. Recent work has focused on the use of immune modulators as a mechanism to facilitate the clearance of HIV in cure strategies.Latently infected cells form a reservoir of persistent HIV despite antiretroviral therapy and host immune responses. Since latency represents the major barrier to eradication of HIV, strategies to activate HIV transcription in latently infected T cells are under investigation. This ‘kick and kill’ strategy involves activation (‘kick’) of HIV replication and a subsequent ‘kill’ by viral cytopathic effect or immune-mediated killing. Early clinical trials using inhibitors of histone deactylase that activate HIV from latency, such as sodium valproate did not show an effect but subsequent work with more potent inhibitors, such as vorinostat,panabinostat and romidepsin have shown that virus replication can be demonstrated in patients on therapy but this replication is not associated with significant increase in plasma HIV viral load or decrease in viral DNA reservoir. This may reflect changes in the CTL killing in the presence of HDACi or lack of activity of the current immune-based therapies. The range of latency reversing agents (LRA) has expanded rapidly with the development of in vitro and ex vivo models of latency that can be used to test the efficacy of these agents in virus reactivation. Use of cytokines, such as type I IFNs, IL-2 or IL-7, may potentially lead to reduction in reservoir size but larger deﬁnitive studies are still needed. Strategies to improve killing of HIV-infected cells have focused on the use of therapeutic vaccination including DC vaccines, the use of cytokines and the use of immune checkpoint blockers such as CTLA-4 and PD-1 that have been shown to be effective in increasing immune responses in cancer therapy.
Despite the efficacy of antiretroviral therapy, immune-based therapies may have a role as adjuvants to antiretroviral therapy for both immune reconstitution and HIV cure. These interventions may augment CD4+ T cell numbers and cell-mediated immunity in the presence of viral suppression with antiretroviral therapy. While the clinical beneﬁts of immune-based therapy remain to be determined, augmentation of antibody-mediated control through potent neutralisation and ADCC, as well as T cell-mediated immune responses through use of immunomodulators, such as immune checkpoint blockers, may play a role to limit viral replication, reduce the risk of drug resistance and eradicate HIV reservoirs in people taking antiretroviral therapy.