Identification of human idiotype-specific T cells in lymphoma and myeloma.

Idiotype protein, among the first identified tumor-specific antigens, has been found to stimulate both humoral and cellular responses in lymphoma and myeloma patients. With the increasing use of B cell depletion treatments such as rituximab in clinic, the cellular response mediated by idiotype-specific T cells has become increasingly important as an adjunct therapy for lymphoma and myeloma. Here, we review the idiotype protein as a tumor antigen and the characteristics of the T cell response elicited idiotype vaccination. We also analyze the T cell epitopes that have been identified in idiotype protein and introduce our new findings of additional T cell epitopes derived from the Ig light chain. Finally, we propose new directions in the generation of idiotype-specific T cells for tumor therapy.

BiovaxID: a personalized therapeutic cancer vaccine for non-Hodgkin's lymphoma.

The clonal immunoglobulin molecule, idiotype (ID), expressed on the surface of B-cell malignancies can function as a tumor-specific antigen. BiovaxID is a patient-specific therapeutic cancer vaccine composed of the tumor idiotype conjugated to a carrier protein, keyhole limpet hemocyanin (KLH). In a Phase II clinical trial, administration of ID-KLH vaccine together with granulocyte-macrophage colony-stimulating factor to follicular lymphoma patients in complete remission induced tumor-specific cellular and humoral immunity and molecular remissions, and was associated with prolonged disease-free survival. A randomized, double-blind, Phase III clinical trial is ongoing to definitively determine the clinical benefit of BiovaxID plus granulocyte-macrophage colony-stimulating factor vaccination in patients with follicular lymphoma.

Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3- cotransporter.

The NBC1 Na+/HCO3- cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO3- absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pH(i)) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pH(i) regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and HCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon.

Therapeutic lymphoma vaccines: importance of T-cell immunity.

The unique antigenic determinants, termed idiotype, of the immunoglobulin expressed on a given B-cell malignancy can serve as a tumor-specific antigen for active immunotherapy. Administration of autologous tumor-derived idiotype protein conjugated to a carrier protein, keyhole limpet hemocyanin, together with granulocyte-macrophage colony-stimulating factor to follicular lymphoma patients in complete clinical remission was associated with induction of tumor-specific cellular and humoral immunity, molecular remissions, and prolonged disease-free survival. Idiotype vaccination in patients with mantle cell lymphoma following rituximab-containing chemotherapy induced tumor-specific T-cell immunity in the absence of B cells, suggesting that vaccines may be used in combination with rituximab. Three double-blind, randomized, Phase III idiotype vaccine trials are currently ongoing to definitively determine the clinical benefit of idiotype-keyhole limpet hemocyanin plus granulocyte-macrophage colony-stimulating factor vaccination in patients with lymphoma. Results from early clinical trials with idiotype vaccines suggested that both humoral and cellular immune responses may be independently associated with tumor regression and improved progression-free survival. With the increased use of rituximab for the treatment of follicular lymphoma and other B-cell non-Hodgkin's lymphomas, further improvement in the potency of the vaccines would require strategies to enhance T-cell responses, as rituximab depletes normal B cells and impairs the generation of antibody responses.

Blood pressure maintenance in NHE3-deficient mice with transgenic expression of NHE3 in small intestine.

NHE3 Na(+)/H(+) exchanger knockout (Nhe3(-/-)) mice have severe absorptive deficits in the kidney proximal tubule and intestinal tract. The resulting hypovolemia has confounded efforts to carefully evaluate the specific effects of NHE3 deficiency on kidney function. Development of mice with transgenic expression of NHE3 in the small intestine (tgNhe3(-/-)) has allowed us to analyze the role of renal NHE3 in overall maintenance of blood pressure, pressure natriuresis, and autoregulation of both glomerular filtration rate (GFR) and renal blood flow (RBF). Ambulatory blood pressure, measured by telemetry, was lower in tgNhe3(-/-) mice than in wild-type controls (tgNhe3(+/+)) when the mice were maintained on a normal NaCl diet but was normalized when they were provided with a high NaCl intake. Furthermore, administration of the AT1-receptor blocker losartan showed that circulating ANG II plays a major role in maintaining blood pressure in tgNhe3(-/-) mice fed normal NaCl but not in those receiving high NaCl. Clearance studies revealed a blunted pressure-natriuresis response in tgNhe3(-/-) mice at lower blood pressures but a robust response at higher blood pressures. Autoregulation of GFR and RBF was normal in tgNhe3(-/-) mice. These results show that dietary NaCl loading normalizes blood pressure in awake tgNhe3(-/-) mice and that alterations in NHE3 activity are not essential for normal autoregulation of GFR and RBF. Furthermore, the data strongly support the hypothesis that NHE3 plays an important role in the diuretic and natriuretic responses to increases in blood pressure but also show that mechanisms not involving NHE3 mediate pressure natriuresis in the higher range of blood pressures studied.

Mice with a targeted disruption of the AE2 Cl-/HCO3- exchanger are achlorhydric.

The AE2 Cl-/HCO3- exchanger is expressed in numerous cell types, including epithelial cells of the kidney, respiratory tract, and alimentary tract. In gastric epithelia, AE2 is particularly abundant in parietal cells, where it may be the predominant mechanism for HCO3- efflux and Cl- influx across the basolateral membrane that is needed for acid secretion. To investigate the hypothesis that AE2 is critical for parietal cell function and to assess its importance in other tissues, homozygous null mutant (AE2(-/-)) mice were prepared by targeted disruption of the AE2 (Slc4a2) gene. AE2(-/-) mice were emaciated, edentulous (toothless), and exhibited severe growth retardation, and most of them died around the time of weaning. AE2(-/-) mice exhibited achlorhydria, and histological studies revealed abnormalities of the gastric epithelium, including moderate dilation of the gastric gland lumens and a reduction in the number of parietal cells. There was little evidence, however, that parietal cell viability was impaired. Ultrastructural analysis of AE2(-/-) gastric mucosa revealed abnormal parietal cell structure, with severely impaired development of secretory canaliculi and few tubulovesicles but normal apical microvilli. These results demonstrate that AE2 is essential for gastric acid secretion and for normal development of secretory canalicular and tubulovesicular membranes in mouse parietal cells.

Functional roles of the alpha isoforms of the Na,K-ATPase.

The Na,K-ATPase is composed of two subunits, alpha and beta, and each subunit consists of multiple isoforms. In the case of alpha, four isoforms, alpha1, alpha2, alpha3, and alpha4 are present in mammalian cells. The distribution of these isoforms is tissue- and developmental-specific, suggesting that they may play specific roles, either during development or coupled to specific physiological processes. In order to understand the functional properties of each of these isoforms, we are using gene targeting, where animals are produced lacking either one copy or both copies of the corresponding gene or have a modified gene. To date, we have produced animals lacking the alpha1 and alpha2 isoform genes. Animals lacking both copies of the alpha1 isoform gene are not viable, while animals lacking both copies of the alpha2 isoform gene make it to birth, but are either born dead or die very soon after. In the case of animals lacking one copy of the alpha1 or alpha2 isoform gene, the animals survive and appear healthy. Heart and EDL muscle from animals lacking one copy of the alpha2 isoform exhibit an increase in force of contraction, while there is reduced force of contraction in both muscles from animals lacking one copy of the alpha1 isoform gene. These studies indicate that the alpha1 and alpha2 isoforms carry out different physiological roles. The alpha2 isoform appears to be involved in regulating Ca(2+) transients involved in muscle contraction, while the alpha1 isoform probably plays a more generalized role. While we have not yet knocked out the alpha3 or alpha4 isoform genes, studies to date indicate that the alpha4 isoform is necessary to maintain sperm motility. It is thus possible that the alpha2, alpha3, and alpha4 isoforms are involved in specialized functions of various tissues, helping to explain their tissue- and developmental-specific regulation.

Renal function in NHE3-deficient mice with transgenic rescue of small intestinal absorptive defect.

The degree to which loss of the NHE3 Na(+)/H(+) exchanger in the kidney contributes to impaired Na(+)-fluid volume homeostasis in NHE3-deficient (Nhe3(-/-)) mice is unclear because of the coexisting intestinal absorptive defect. To more accurately assess the renal effects of NHE3 ablation, we developed a mouse with transgenic expression of rat NHE3 in the intestine and crossed it with Nhe3(-/-) mice. Transgenic Nhe3(-/-) (tgNhe3(-/-)) mice tolerated dietary NaCl depletion better than nontransgenic knockouts and showed no evidence of renal salt wasting. Unlike nontransgenic Nhe3(-/-) mice, tgNhe3(-/-) mice tolerated a 5% NaCl diet. When fed a 5% NaCl diet, tgNhe3(-/-) mice had lower serum aldosterone than tgNhe3(-/-) mice on a 1% NaCl diet, indicating improved extracellular fluid volume status. Na(+)-loaded tgNhe3(-/-) mice had sharply increased urinary Na(+) excretion, reflective of increased absorption of Na(+) in the small intestine; nevertheless, they remained hypotensive, and renal studies showed a reduction in glomerular filtration rate (GFR) similar to that observed in nontransgenic Nhe3(-/-) mice. These data show that reduced GFR, rather than being secondary to systemic hypovolemia, is a major renal compensatory mechanism for the loss of NHE3 and indicate that loss of NHE3 in the kidney alters the set point for Na(+)-fluid volume homeostasis.

In vivo evidence for interferon-gamma-mediated homeostatic mechanisms in small intestine of the NHE3 Na+/H+ exchanger knockout model of congenital diarrhea.

Mice lacking NHE3, the major absorptive Na(+)/H(+) exchanger in the intestine, are the only animal model of congenital diarrhea. To identify molecular changes underlying compensatory mechanisms activated in chronic diarrheas, cDNA microarrays and Northern blot analyses were used to compare global mRNA expression patterns in small intestine of NHE3-deficient and wild-type mice. Among the genes identified were members of the RegIII family of growth factors, which may contribute to the increased absorptive area, and a large number of interferon-gamma-responsive genes. The latter finding is of particular interest, since interferon-gamma has been shown to regulate ion transporter activities in intestinal epithelial cells. Serum interferon-gamma was elevated 5-fold in NHE3-deficient mice; however, there was no evidence of inflammation, and unlike conditions such as inflammatory bowel disease, levels of other cytokines were unchanged. In addition, quantitative PCR analysis showed that up-regulation of interferon-gamma mRNA was localized to the small intestine and did not occur in the colon, spleen, or kidney. These in vivo data suggest that elevated interferon-gamma, produced by gut-associated lymphoid tissue in the small intestine, is part of a homeostatic mechanism that is activated in response to the intestinal absorptive defect in order to regulate the fluidity of the intestinal tract.

Roles of the Na,K-ATPase alpha4 isoform and the Na+/H+ exchanger in sperm motility.

The Na,K-ATPase generates electrochemical gradients that are used to drive the coupled transport of many ions and nutrients across the plasma membrane. The functional enzyme is comprised of an alpha and beta subunit and families of isoforms for both subunits exist. Recent studies in this laboratory have identified a biological role for the Na,K-ATPase alpha4 isoform in sperm motility. Here we further investigate the role of the Na,K-ATPase carrying the alpha4 isoform, showing again that ouabain eliminates sperm motility, and in addition, that nigericin, a H+/K+ ionophore, and monensin, a H+/Na+ ionophore, reinitiate motility. These data, along with the observation that the K+ ionophore valinomycin has no effect on the motility of ouabain-inhibited sperm, suggest that ouabain may change intracellular H+ levels in a manner that is incompatible with sperm motility. We have also localized NHE1 and NHE5, known regulators of intracellular H+ content, to the same region of the sperm as the Na,K-ATPase alpha4 isoform. These data highlight the important role of the Na,K-ATPase alpha4 isoform in regulating intracellular H(+) levels, and provide evidence suggesting the involvement of the Na+/H+ exchanger, which is critical for maintaining normal sperm motility.

Intestinal NaCl transport in NHE2 and NHE3 knockout mice.

Sodium/proton exchangers [Na(+)/H(+) (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na(+) and Cl(-) flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(-)], and NHE3 knockout [NHE3(-)] mice. Under basal conditions, NHE(+) and NHE2(-) jejuna had similar rates of net Na(+) (approximately 6 microeq/cm(2) x h) and Cl(-) (approximately 3 microeq/cm(2) x h) absorption. In contrast, NHE3(-) jejuna had reduced net Na(+) absorption (approximately 2 microeq/cm(2) x h) but absorbed Cl(-) at rates similar to NHE(+) and NHE2(-) jejuna. Treatment with 100 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA) completely inhibited net Na(+) and Cl(-) absorption in all genotypes. Studies of the Na(+) absorptive flux (J) indicated that J in NHE(+) jejunum was not sensitive to 1 microM EIPA, whereas J in NHE3(-) jejunum was equally sensitive to 1 and 100 microM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMP(i)) abolished net NaCl absorption and stimulated electrogenic Cl(-) secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(-) and NHE3(-) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na(+) absorption; 2) an amiloride-sensitive Na(+) transporter partially compensates for Na(+) absorption in NHE3(-) jejunum; 3) cAMP(i) stimulation abolishes net Na(+) absorption in NHE(+), NHE2(-), and NHE3(-) jejunum; and 4) electroneutral Cl(-) absorption is not directly dependent on either NHE2 or NHE3.