Claire W. Hallahan

HIV-specific CD8+ T cell proliferation is coupled to perforin expression and is maintained in nonprogressors

It is unclear why immunological control of HIV replication is incomplete in most infected individuals. We examined here the CD8+ T cell response to HIV-infected CD4+ T cells in rare patients with immunological control of HIV. Although high frequencies of HIV-specific CD8+ T cells were present in nonprogressors and progressors, only those of nonprogressors maintained a high proliferative capacity. This proliferation was coupled to increases in perforin expression. These results indicated that nonprogressors were differentiated by increased proliferative capacity of HIV-specific CD8+ T cells linked to enhanced effector function. In addition, the relative absence of these functions in progressors may represent a mechanism by which HIV avoids immunological control.

Cyclophosphamide-induced cystitis and bladder cancer in patients with Wegener granulomatosis.

Wegener granulomatosis is a necrotizing granulomatous vasculitis that typically involves the upper and lower respiratory tract and the kidneys. Standard therapy for Wegener granulomatosis includes the daily administration of low-dose oral cyclophosphamide and corticosteroid therapy [1, 2]. This therapeutic regimen has dramatically improved the survival of patients with this otherwise fatal disease: More than 90% of patients treated with cyclophosphamide and corticosteroid therapy improve markedly, and 75% achieve complete remission of disease [1, 3]. However, extended follow-up of patients with Wegener granulomatosis indicates that relapse of disease is common, and repeated and prolonged courses of cyclophosphamide can be associated with serious long-term toxicities, including bone marrow suppression, infertility, hemorrhagic cystitis, and the development of cancer [3]. Hemorrhagic cystitis and bladder cancer are well-recognized complications of cyclophosphamide therapy for both malignant [4] and nonmalignant diseases [3, 5-7]. However, the relations among total cyclophosphamide dose, the development of cystitis, and the occurrence of bladder cancer have not been well defined. In this report, we describe the incidence, clinical manifestations, and natural history of cyclophosphamide-mediated urotoxicity in a cohort of patients with Wegener granulomatosis. We identify risk factors associated with the development of cyclophosphamide-induced bladder cancer and discuss recommendations for surveillance. Methods Patients From 1967 to 1993, 145 patients with Wegener granulomatosis were treated with cyclophosphamide at the Warren G. Magnuson Clinical Center of the National Institutes of Health (NIH). The clinical features of the underlying disease in all but 3 of these patients have been reported previously [3]; the clinical and demographic characteristics of these patients are summarized in Table 1. Individual patients were followed for 0.5 to 27 years (median, 8.5 years), for a total of 1333 patient-years. Table 1. Demographic and Clinical Characteristics of 145 Patients with Wegener Granulomatosis Who Were Treated with Cyclophosphamide* Treatment Protocol We used the standard cyclophosphamide treatment regimen, which has been described previously [1, 3]. Therapy consisted of 1) oral cyclophosphamide, 2 mg/kg of body weight per day and 2) prednisone, 1 mg/kg of body weight per day. If patients improved substantially after the first month of treatment, the prednisone dose was gradually tapered to an alternate-day regimen, and prednisone therapy was eventually discontinued. Cyclophosphamide therapy was continued for at least 1 year after patients achieved complete remission. The cyclophosphamide dose was then tapered by 25-mg decrements of therapy every 2 to 3 months until discontinuation of therapy or until disease recurrence required an increase in dose. The cyclophosphamide dose was adjusted as needed to keep the absolute neutrophil count higher than 1.0 106/L. If substantial toxicity required the permanent discontinuation of cyclophosphamide therapy and if signs of active vasculitis were present, azathioprine, chlorambucil, or (after 1990) low-dose weekly methotrexate therapy was allowed. Eight patients with fulminant disease initially received intravenous cyclophosphamide at daily doses of 3 to 5 mg/kg. When their disease stabilized, these patients were switched to the standard oral cyclophosphamide regimen. Fourteen patients received monthly pulses of intravenous cyclophosphamide, 1 g/m2 body surface area; 13 of these 14 patients also received one or more courses of the standard oral cyclophosphamide regimen. Thus, 144 of the 145 patients received oral cyclophosphamide therapy for some period of time. Patients were evaluated at the NIH every 1 to 3 months. Those who achieved remission of disease and maintained it for 1 year were subsequently seen every 6 months. For each patient, urinalysis was done at every visit, and a cytologic examination of urine was done every 6 to 12 months. All patients received cyclophosphamide as part of clinical research protocols approved by the National Institute of Allergy and Infectious Diseases (NI-AID) Institutional Review Board, the NIAID Clinical Director, and the director of the NIH Clinical Center. All patients gave written informed consent. Urine Cytology Cytologic examination of urine was done at each evaluation. Sediments obtained from voided urine specimens were fixed in Saccomanno solution (Lerner Laboratories, Pittsburgh, Pennsylvania), immobilized on membrane filters (Millipore, Chicago, Illinois) or by cytospin, placed in 95% ethanol, and stained with Papanicolaou stain. Urine samples obtained as much as 6 months before each cystoscopic examination were reviewed retrospectively and were correlated with subsequent bladder biopsy specimens. Cellular cytologic features were placed in the following categories according to the following criteria [8-10]: 1. Negative: no important epithelial abnormalities. 2. Atypia: some nuclear abnormalities in epithelial cells, but the changes could not be definitely placed in categories 3, 4, or 5. 3. Therapeutic or viral: cytologic changes consistent with polyomavirus infection or cyclophosphamide toxicity. In the absence of diagnostic inclusions, the two types of changes are indistinguishable, and we therefore grouped them together. The nuclear enlargement and hyperchromasia associated with polyomavirus or chemotherapeutic effect should be distinguished from high-grade dysplasia or carcinoma. 4. Dysplasia or possible low-grade transitional-cell carcinoma: a few cells in a voided urine sample, either singly or in clusters, that have slightly enlarged, irregular nuclei with increased granularity in chromatin distribution and small or absent nucleoli. 5. Transitional-cell carcinoma: high-grade lesions meeting unequivocal criteria of malignancy. Definition of Terms Nonglomerular hematuria was defined as microscopic or gross hematuria not associated with the presence of erythrocyte casts or declining renal function. Glomerulonephritis causing hematuria associated with erythrocyte casts (glomerular hematuria) occurred at least once in 116 of the 145 patients (Table 1). If hematuria persisted after the treatment of glomerulonephritis and the disappearance of erythrocyte casts, or if hematuria not associated with the presence of erythrocyte casts ever developed, patients were considered to have nonglomerular hematuria and had cystoscopy (see below). Cyclophosphamide-induced cystitis was defined as nonglomerular hematuria associated with characteristic cystoscopic bladder changes. These changes included patchy areas of neovascularity and telangiectasia manifested as an increased number of tortuous, thin-walled veins and small areas of hemorrhage in or under the bladder epithelium. The mucosa between the hypervascular areas may appear normal or pale with decreased vascularity. Cystoscopy Cystoscopy was done to evaluate nonglomerular hematuria (microscopic or gross) in patients receiving cyclophosphamide. Only the results of cystoscopies done at the NIH Clinical Center by members of the Urologic Oncology Section of the National Cancer Institute are included in this report. Most of these cystoscopies were done by two of the authors; all cystoscopy results were reviewed by these two authors. Random biopsies were done only if the results of cytologic examination of urine suggested malignancy. Patients having cystoscopy had intravenous pyelography or retrograde pyelography, or both, at least once to evaluate the upper urinary tract. Statistical Analysis The frequencies of clinical findings were compared by using the Fisher exact test for association with bladder cancer and nonglomerular hematuria; adjustments were made for multiple comparisons of microscopic and gross hematuria with bladder cancer using the modified Bonferroni method [11]. The effects of fixed covariates (sex, history of smoking, age at onset of Wegener granulomatosis disease, and duration of disease before first cyclophosphamide treatment) and time-varying covariates (microscopic hematuria, gross hematuria, total cyclophosphamide dose, and duration of cyclophosphamide therapy) on the development of bladder cancer were examined using Cox proportional-hazards regression analysis [12, 13]. Medians and other percentiles for variables dependent on follow-up time were estimated by using the Kaplan-Meier method [14]. The cumulative distributions determined by the Kaplan-Meier method were compared with the log-rank test. Risk estimates for the development of bladder cancer in patients with Wegener granulomatosis who were treated with cyclophosphamide were determined by comparing observed rates with the expected rates for the United States population, which were obtained from the Surveillance, Epidemiology, and End Results (SEER) Cancer Statistics Review, 1973-1991 [15]. Results Hematuria Seventy-three of 145 patients treated with cyclophosphamide (50%) developed nonglomerular hematuria; the median time to development for all patients was 37 months of receipt of cyclophosphamide (95% CI, 32 to 55 months), and the median dose before development was 124 g (CI, 82 to 149 g) (Table 2 and Figure 1). Forty-one patients (56%) presented with microscopic hematuria; 32 (44%) presented with gross hematuria. Twenty-eight patients had more than one recurrent episode of nonglomerular hematuria (microscopic or gross), sometimes years after cyclophosphamide therapy had been discontinued. Eighteen of the 73 patients developed hematuria after cyclophosphamide therapy was discontinued. Manifestations of active vasculitis necessitated that cyclophosphamide therapy be continued in 26 of the 55 patients who developed nonglomerular hematuria while receiving this therapy. Table 2. Clinical Characteristics of the 73 Patients Treated with Cyclophosphamide Who Developed Nonglomerular Hematuria Figure 1. Cumulative r

Randomized, controlled trial of recombinant human α-interferon in patients with chronic hepatitis B

Forty-five patients with chronic hepatitis B were entered into a randomized controlled trial of recombinant human alpha-interferon therapy. All patients had hepatitis B surface antigen in serum for at least 1 yr and had stable serum levels of both hepatitis B virus deoxyribonucleic acid and hepatitis B e antigen. During the 4-mo period of therapy, 10 of 31 (32%) treated patients and only 1 of 14 (7%) control patients became negative for serum hepatitis B virus deoxyribonucleic acid and deoxyribonucleic acid polymerase. All 10 patients who became negative for serum hepatitis B virus deoxyribonucleic acid subsequently had a marked improvement in serum aminotransferase activities and lost hepatitis B e antigen from serum, and 9 of them had improvement in liver histology. Comparison of responders to nonresponders indicated that female sex and a high initial level of serum aspartate aminotransferase correlated best with response to interferon therapy. These findings indicate that a 4-mo course of recombinant alpha-interferon can induce a remission in disease in approximately one-third of patients with chronic hepatitis B.

Lytic Granule Loading of CD8+ T Cells Is Required for HIV-Infected Cell Elimination Associated with Immune Control

Virus-specific CD8+ T cells probably mediate control over HIV replication in rare individuals, termed long-term nonprogressors (LTNPs) or elite controllers. Despite extensive investigation, the mechanisms responsible for this control remain incompletely understood. We observed that HIV-specific CD8+ T cells of LTNPs persisted at higher frequencies than those of treated progressors with equally low amounts of HIV. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This function required lytic granule loading of effectors and delivery of granzyme B to target cells. Defective cytotoxicity of progressor effectors could be restored after treatment with phorbol ester and calcium ionophore. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. They also demonstrate that lytic granule contents of memory cells are a critical determinant of cytotoxicity that must be induced for maximal per-cell killing capacity.

Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1-infected patients receiving highly active anti-retroviral therapy

The size of the pool of resting CD4+ T cells containing replication-competent HIV in the blood of patients receiving intermittent interleukin (IL)-2 plus highly active anti-retroviral therapy (HAART) was significantly lower than that of patients receiving HAART alone. Virus could not be isolated from the peripheral blood CD4+ T cells in three patients receiving IL-2 plus HAART, despite the fact that large numbers of resting CD4+ T cells were cultured. Lymph node biopsies were done in two of these three patients and virus could not be isolated. These results indicate that the intermittent administration of IL-2 with continuous HAART may lead to a substantial reduction in the pool of resting CD4+ T cells that contain replication-competent HIV.

Persistence of HIV in Gut-Associated Lymphoid Tissue despite Long-Term Antiretroviral Therapy

Human immunodeficiency virus (HIV) persists in peripheral blood mononuclear cells despite sustained, undetectable plasma viremia resulting from long-term antiretroviral therapy. However, the source of persistent HIV in such infected individuals remains unclear. Given recent data suggesting high levels of viral replication and profound depletion of CD4(+) T cells in gut-associated lymphoid tissue (GALT) of animals infected with simian immunodeficiency virus and HIV-infected humans, we sought to determine the level of CD4(+) T cell depletion as well as the degree and extent of HIV persistence in the GALT of infected individuals who had been receiving effective antiviral therapy for prolonged periods of time. We demonstrate incomplete recoveries of CD4(+) T cells in the GALT of aviremic, HIV-infected individuals who had received up to 9.9 years of effective antiretroviral therapy. In addition, we demonstrate higher frequencies of HIV infection in GALT, compared with PBMCs, in these aviremic individuals and provide evidence for cross-infection between these 2 cellular compartments. Together, these data provide a possible mechanism for the maintenance of viral reservoirs revolving around the GALT of HIV-infected individuals despite long-term viral suppression and suggest that the GALT may play a major role in the persistence of HIV in such individuals.

Maintenance of Large Numbers of Virus-Specific CD8+ T Cells in HIV-Infected Progressors and Long-Term Nonprogressors

The virus-specific CD8+ T cell responses of 21 HIV-infected patients were studied including a unique cohort of long-term nonprogressors with low levels of plasma viral RNA and strong proliferative responses to HIV Ags. HIV-specific CD8+ T cell responses were studied by a combination of standard cytotoxic T cell (CTL) assays, MHC tetramers, and TCR repertoire analysis. The frequencies of CD8+ T cells specific to the majority of HIV gene products were measured by flow cytometric detection of intracellular IFN-γ in response to HIV-vaccinia recombinant-infected autologous B cells. Very high frequencies (0.8–18.0%) of circulating CD8+ T cells were found to be HIV specific. High frequencies of HIV-specific CD8+ T cells were not limited to long-tern nonprogressors with restriction of plasma virus. No correlation was found between the frequency of HIV-specific CD8+ T cells and levels of plasma viremia. In each case, the vast majority of cells (up to 17.2%) responded to gag-pol. Repertoire analysis showed these large numbers of Ag-specific cells were scattered throughout the repertoire and in the majority of cases not contained within large monoclonal expansions. These data demonstrate that high numbers of HIV-specific CD8+ T cells exist even in patients with high-level viremia and progressive disease. Further, they suggest that other qualitative parameters of the CD8+ T cell response may differentiate some patients with very low levels of plasma virus and nonprogressive disease.

Frequency and Phenotype of Human Immunodeficiency Virus Envelope-Specific B Cells from Patients with Broadly Cross-Neutralizing Antibodies

ABSTRACT Induction of broadly cross-reactive neutralizing antibodies (NAb) is an important goal for a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. Some HIV-infected patients make a NAb response that reacts with diverse strains of HIV-1, but most candidate vaccines have induced NAb only against a subset of highly sensitive isolates. To better understand the nature of broad NAb responses that arise during natural infection, we screened patients for sera able to neutralize diverse HIV strains and explored the frequency and phenotype of their peripheral Envelope-specific B cells. We screened 113 HIV-infected patients of various clinical statuses for the prevalence of broad NAb. Sera able to neutralize at least four of five viral isolates were found in over one-third of progressors and slow progressors, but much less frequently in aviremic long-term nonprogressors. Most Env-specific antibody-secreting B cells were CD27hi CD38hi plasmablasts, and the total plasmablast frequency was higher in HIV-infected patients than in uninfected donors. We found that 0.0031% of B cells and 0.047% of plasmablasts secreted Env-specific immunoglobulin G (IgG) in an enzyme-linked immunospot (ELISPOT) assay. We developed a novel staining protocol to label HIV-specific B cells with Env gp140 protein. A total of 0.09% of B cells were found to be Env-specific by this method, a frequency far higher than that indicated by ELISPOT assay. gp140-labeled B cells were predominantly CD27+ and surface IgG+. These data describe the breadth and titer of serum NAb and the frequency and phenotype of HIV-specific B cells in a cohort of patients with broad cross-neutralizing antibody responses that are potential goals for vaccines for HIV.

HIV-1 induces phenotypic and functional perturbations of B cells in chronically infected individuals

A number of perturbations of B cells has been described in the setting of HIV infection; however, most remain poorly understood. To directly address the effect of HIV replication on B cell function, we investigated the capacity of B cells isolated from HIV-infected patients to respond to a variety of stimuli before and after reduction of viremia by effective antiretroviral therapy. B cells taken from patients with high levels of plasma viremia were defective in their proliferative responses to various stimuli. Viremia was also associated with the appearance of a subpopulation of B cells that expressed reduced levels of CD21. After fractionation into CD21high- and CD21low-expressing B cells, the CD21low fraction showed dramatically reduced proliferation in response to B cell stimuli and enhanced secretion of immunoglobulins when compared with the CD21high fraction. Electron microscopic analysis of each fraction revealed cells with plasmacytoid features in the CD21low B cell population but not in the CD21high fraction. These results indicate that HIV viremia induces the appearance of a subset of B cells whose function is impaired and which may be responsible for the hypergammaglobulinemia associated with HIV disease.

HIV-infected individuals receiving effective antiviral therapy for extended periods of time continually replenish their viral reservoir

The persistence of latently infected, resting CD4+ T cells is considered to be a major obstacle in preventing the eradication of HIV-1 even in patients who have received effective antiviral therapy for an average duration of 5 years. Although previous studies have suggested that the latent HIV reservoir in the resting CD4+ T cell compartment is virologically quiescent in the absence of activating stimuli, evidence has been mounting to suggest that low levels of ongoing viral replication persist and in turn, prolong the overall half-life of HIV in patients receiving antiviral therapy. Here, we demonstrate the persistence of replication-competent virus in CD4+ T cells in a cohort of patients who had received uninterrupted antiviral therapy for up to 9.1 years that rendered them consistently aviremic throughout that time. Surprisingly, substantially higher levels of HIV proviral DNA were found in activated CD4+ T cells when compared with resting CD4+ T cells in the majority of patients we studied. Phylogenetic analyses revealed evidence for cross infection between the resting and activated CD4+ T cell compartments, suggesting that ongoing reactivation of latently infected, resting CD4+ T cells and spread of virus by activated CD4+ T cells may occur in these patients. Such events may allow continual replenishment of the CD4+ T cell reservoir and resetting of the half-life of the latently infected, resting CD4+ T cells despite prolonged periods of aviremia.