Synergistic effects of human B and T lymphocyte mitogens induce uniform,high levels of immunoglobulin secretion among normal donors

Synergistic effects of B-cell mitogen Staphylococcus bacteria strain Cowan I (Cowan I) plus T-cell activator pokeweed mitogen (PWM) in generating immunoglobulin-secreting cells (ISC) from human peripheral blood mononuclear cells (MNC) were investigated. ISC were assayed by reverse plaque-forming cells using protein A-coated red blood cells. Low concentrations of PWM plus Cowan I gave superadditive effects on ISI induction, generating 3–20 times as many ISC as optimal amounts of either mitogen alone. The mitogens together and separately showed similar kinetics of ISC; synergy was observed at every day tested. IgM ISC represented 10% of initial MNC from all cell donors tested even if the donors were low responders to either mitogen alone. The numbers of ISC obtained are higher than previous reports and more uniform among donors, making this a superlative method for studies on normal human immunoglobulin secretion.     

Human IgM+IgD+ B cells,the major B cell subset in the peripheral blood,express Vϰ genes with no or little somatic mutation throughout life

Peripheral blood B cells of a 67-year-old person were separated into IgM⁺IgD⁺, IgM⁺IgD^?, and IgM^?IgD^? subsets, and nucleotide sequences of expressed immunoglobulin light chain variable (V) regions encoded by V?3 and V?4 gene family members were determined from amplified cDNA. V region sequences from IgM⁺IgD⁺ cells (the major B cell population in the blood) showed no or little somatic mutation (0.3%), in contrast to V? sequences from IgM⁺IgD^? and IgM^?IgD^? B cells (2.0% and 3.9%, respectively). This suggests that in the human like in the mouse, and independently of age, somatically mutated memory B cells accumulate in the compartment of IgM^?IgD^? cells, whereas the IgM⁺IgD⁺ subpopulation consists of cells whose antibody repertoire is mainly determined by V region gene rearrangements and N-region insertion, at the molecular level. The somatically mutated IgM+IgD^? cells may represent early descendants of IgM⁺IgD⁺ cells recruited into the memory cell compartment.     

Functional Maturation of Immature b Cells Accumulated in the Periphery by an Intraperitoneal Administration of a Traditional Chinese Medicine,Xiao-Chai-Hu-Tang (Japanese Name: Shosaiko-To)

Abstract

We found that an intraperitoneal (ip) injection of a traditional Chinese herbal medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to), induced accumulation of B lymphocytes (sIgM⁺) in the peritoneal cavity and spleen. 1) Cell surface marker analysis by a fluorescence-activated cell sorter (FACS) demonstrated that the accumulated B cells on day 4 or 7 after shosaiko-to administration (early phase) were composed mainly of sIgM⁺ IgD^- cells and suggested that these B cells maturated into sIgM⁺ IgD⁺ cells on days 10 or 14 (late phase). Relative decrease of IgM⁺ IgD⁺cells at early phase was more profound in peritoneal cells (PC) than in spleen cells. 2) With respect to spleen lymphocytes, antibody responses to a thymus-independent (TI) antigen of type 2 (trinitrophenylated Ficoll) and a thymus-dependent (TD) antigen (sheep erythrocyte) were enhanced at late phase but not at early phase. In contrast, responses to trinitrophenylated lipopolysaccharide (TNP-LPS) as a TI-1 antigen and LPS as a B cell mitogen or a polyclonal B cell activator were enhanced markedly at early phase but declined at late phase. 3) With respect to peritoneal lymphocytes, responses to LPS were suppressed at early phase but recovered at late phase. Enhanced responses to TI and TD antigen at late phase in spleen lymphocytes and suppressed response to LPS at early phase in peritoneal lymphocytes may be explained by increases of IgM⁺ IgD⁺ mature B cells and IgM⁺ IgD^- immature B cells, respectively, at those times. Enhanced responses to TI-1 or LPS in spleen lymphocytes at early phase may be explained by elevated sensitivity of IgM⁺ IgD⁺ cells which reside in the spleen before shosaiko-to administration and receive the direct stimulation by shosaiko-to, or by acquired responsiveness of IgM⁺ IgD^- cells which migrate after stimulation with shosaiko-to.     

Is the activated T helper stimulus able to induce Ig isotype switching in cultured human B cells?

It Is confirmed that large amounts of IgM, IgG, and IgA areproduced when human B cells are cultured with T cells activatedby immobilized CD3 antibody (CD3 system). IL-2 was essential;lowerlevels of Ig production with different isotype ratios were obtainedif IL-4 or IL-6 replaced IL-2. Depletion of sIgG⁺ or sIgA⁺ cellsfrom the B population to be cultured markedly reduced productionof IgG or IgA. Culturesof B cells selected with the pan-B markersCD19, CD72, or CD21 contained similar levels of Ig of all threeisotypes, whereas B cells selected for sIgM or sIgD expressionproduced IgM but very little IgG or IgA indicating that littleisotype switching was occurring. Production of IgG or IgA fromcells expressing these isotypes was more efficient than productionof IgM from IgM⁺ IgD⁺ cells. These results are considered inthe light of the demonstration by others of the production ofmultiple isotypes from single sIgM⁺-selected B cells. Clonedhuman T cells from a single donor induced production of allthree isotypes, but the proportions varied indicating that thepotent T-B cell interactions inducing B cell activation mayoverride and conceal the operation of isotype specific cellinteractions. Some T clones used at an optimal dose were aseffective untreated as X-irradiated, whereas with other clonesmaximumIg production was not achieved without irradiation.     

B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B cells and helper T cells in immunoglobulin production

B cell immunoglobulin production is regulated by helper T cells through direct interaction and secreted cytokines. In the present study, we functionally analyzed CD27 in cord and peripheral blood B cells. Adult peripheral blood B cells were separated into CD27⁺ and CD27^? cells, which differed in their morphology. Cord blood B cells did not express CD27, and CD27 expression on peripheral blood B cells increased with age. Only CD27⁺ B cells had the ability to produce immunoglobulin, which was increased by contact with a tumor necrosis factor-related transmembrane ligand, CD70. Adult peripheral blood CD27⁺ B cells can be further subdivided into two discrete subtypes: IgD^?CD27⁺ and IgD⁺ CD27⁺ B cells. IgD^? CD27⁺ B cells produce IgG, IgM and IgA, whereas IgD⁺ CD27⁺ B cells predominantly produce IgM. The addition of activated CD4⁺ CD45RO T cells expressing CD70 caused down-regulation of CD27 expression on activated B cells, and this down-modulation was completely blocked by anti-CD70 monoclonal antibody, indicating direct T-B cell contact via CD27/CD70. The triggering via CD27 and CD40 additively increased the immunoglobulin production under Staphylococcus aureus Cowan strain plus interleukin-2 stimulation. Taken together, our findings demonstrate that peripheral blood B cells are separated into subpopulations by CD27 and IgD expression and that CD27⁺ B cells produce large amounts of immunoglobulin by interaction with the CD70 molecule.     

Spontaneous immunoglobulin-producing capacity of cultures from lupus patients and normal donors following depletion of cells expressing CD19 or CD38

Cells spontaneously secreting IgG or IgM (ISC) are present at a high level in the blood of patients with systemic lupus erythematosus (SLE). By use of magnetic-bead techniques, mononuclear cells from such patients and healthy donors were fractionated according to expression of CD19 or CD38 and the cell fractions were then cultured in the absence of added mitogen/antigen for 5/6 days. Supernatant IgG and IgM were determined and, in addition, in the CD38 experiments ISC were enumerated both before and after culture. Much of the immunoglobulin-producing capacity of unfractionated cells (UFC) from both donor groups was recovered in the CD19^? fraction, and no immunoglobulin was produced by CD19⁺ cells suggesting, unexpectedly, that ISC were not expressing CD19. By contrast, CD38 fractionation resulted in nearly all ISC passing to the CD38⁺ fraction which produced levels of immunoglobulin approaching 50% that of UFC. On culture of CD38^? cells there was a build up in the number of IgG and IgM ISC, this being particularly striking in the controls with numbers well in excess of those in UFC. Not all these new ISC became CD38⁺, but the maturation process was more efficient in the SLE patients. The possibility is discussed that the spontaneous response in the CD38^? populations is due to removal of CD38⁺ natural killer (NK) cells. Removal of ISC that are present preculture is a helpful initial step in studying ISC generation in the disease.     

B-lymphocyte Subpopulations in Patients with Selective IgA Deficiency

Introduction

Selective deficiency IgA (IgAD) is the most common primary abnormality of immunoglobulin production with unknown pathophysiology. It is genetically related to common variable immunodeficiency (CVID), where besides IgA also IgG and frequently IgM serum levels are decreased. In this study we focused on determination of B-lymphocyte developmental stages and searching for similarities between CVID and IgAD.

Materials and Methods

Using flow cytometry we determined major lymphocyte subpopulations and B-lymphocyte subsets: na?ve (CD27^-IgD⁺), marginal zone cells (CD27⁺IgD⁺), class-switched memory cells (CD27⁺IgD^-), ??double-negative?? B cells (CD27^-IgD^-), transitional cells (IgM⁺⁺CD38⁺⁺), plasmablasts (CD38⁺⁺⁺IgM⁺ or IgM^-), and CD21^lowCD38^low cells in 80 patients with IgAD, 48 patients with CVID, and 80 control persons.

Results

Compared to healthy controls, a decrease in the absolute number and frequency of CD4+ cells (both?P?P?=?0.035) as well as plasmablasts (P?lowCD38^low subset (P?=?0.007) was observed in IgAD patients compared to control persons. No significant differences were observed in the remaining B-cell developmental subsets. A decrease in CD27⁺IgD^- (<0.4% of peripheral blood lymphocytes), frequently observed in CVID patients and also previously reported in IgAD, was found in only five patients (6%) with IgAD, two of them being first-degree relatives of CVID patients.

Conclusion

Our results show a decrease of terminally differentiated B-lymphocyte subsets in patients with IgAD, similar as previously found in patients with CVID, but these results are less expressed than in CVID patients.     

IgD expression on B cells is more efficient than IgM but both receptors are functionally equivalent in up-regulation CD80/CD86 co-stimulatory molecules

The expression and function of IgM and IgD antigen receptors were studied in a series of anti-hen egg lysozyme (HEL) immunoglobulin (Ig)-transgenic mice expressing either IgM alone, IgD alone, or both IgM and IgD. B cell surface expression of IgD was found to be more efficient than that of IgM. Thus antigen receptor density on IgD⁺, IgM^? B cells was twofold higher than on IgM⁺, IgD^? B cells despite the presence of sevenfold lower levels of membrane heavy chain mRNA, and coexpression of IgD with IgM led to almost complete inhibition of surface IgM. In addition, less extensive down-regulation of IgD occurred following exposure to antigen in vitro. When regulation of CD80/CD86 co-stimulatory molecules by surface Ig was examined, up-regulation of the former was initiated at lower antigen concentrations on IgM^?, IgD⁺ compared to IgM⁺, IgD^? B cells. On correcting for antigen receptor density, however, induction of CD80/CD86 by IgM and IgD was comparable. Taken together, these results reinforced the functional similarity of IgM and IgD antigen receptors while at the same time revealing differences in expression which may explain their simultaneous presence on mature B cells.     

Intestinal B cell defects in common variable immunodeficiency

The humoral immune system of the small intestine of 17 patients with common variable immunodeficiency (CVID) was studied by immunohistology using antibodies specific for IgA1,2, IgM, IgG1-4, the J chain and the secretory component (SC). IgA1,2⁺, IgG2⁺ and IgM⁺ lamina propria B cells were totally lacking in 65% (11/17), 41% (7/17) and 18% (3/17) of CVID patients, respectively. One patient exhibited an isolated IgA1 subclass deficiency. The proportion of plasma cells in conventionally stained histological sections of the same intestinal biopsies showed a close correlation with the numbers of IgA⁺ and IgM⁺ cells. Considerable numbers of J chain-synthesizing cells were present in all patients with CVID, indicating the presence of early B cells unable to differentiate into immunoglobulin-producing plasma cells. Most of the patients with intestinal IgA and/or IgM defects strongly expressed the SC in their enterocytes, suggesting an immunoglobulin-independent regulation of the SC. Clinically, only CVID patients with intestinal IgA defects developed intestinal infections with Giardia lambtia, Campylobacter jejuni or Candida albicans. The outcome of in vitro immunoglobulin synthesis assays with peripheral blood lymphocytes did not predict the presence or absence of the respective isotype-producing B cells in the intestinal lamina propria. Thus, immunohistological examinations of intestinal biopsies are required to determine the extent of mucosal immunodeficiency in CVID patients.     

Selective enhancement of human IgE production in vitro by synergy of pokeweed mitogen and mercuric chloride

IgE production in vitro was investigated in cultures of human peripheral blood mononuclear cells (MNC) from non-atopic donors with pokeweed mitogen (PWM), mercuric chloride (HgCl2), or both. PWM alone induced a few IgE immunoglobulin secreting cells (ISC) detected by reverse plaque forming cells (PFC) and many IgG, IgM, and IgA PFC. HgCl2 alone failed to produce significant numbers of ISC of any class. PWM plus HgCl2 caused a selective increase of IgE PFC without affecting IgG, IgM, and IgA PFC. Co-cultures of B cells plus mitomycin C (MMC) treated T cells stimulated by PWM alone produced more IgG, IgM, IgA and IgE PFC than those of B cells plus T cells. However, PWM plus HgCl2 produced significantly more IgE PFC selectively in those cultures. This effect was observed in the cells of most of the donors, but a few donors showed different responses.     

A novel type of B-cell mitogen isolated from juzen-taiho-to (TJ-48), a Japanese traditional medicine

Juzen-taiho-to (TJ-48), a Japanese traditional medicine, is known to have various immunological activities including the induction of B-cell proliferation. We investigated the properties of the pectic polysaccharide fraction of TJ-48 (F-5-2) which is most active in the proliferation of spleen cells. To an extent equal to that of TJ-48, F-5-2 induced the proliferation of B-cells, particularly those holding both sIgM and sIgD. The proliferation induced by F-5-2 was T-cell independent and macrophage dependent. The macrophages could be substituted for a soluble factor(s) secreted from the macrophages but not for IL-1. Generally, B-cell mitogens are known to induce the proliferation of B-cells and subsequently differentiation into plasma cells. However, although F-5-2 induced the B-cell differentiation, it arrested their development in the intermediate stage of the B-cell differentiation. The B-cells induced by F-5-2 produced IgM antibody in response to IL-6 and an antigen (SRBC) but not IgG antibody. F-5-2 induced the expression of IL-6R not only on IgM⁺ and IgG⁺ B-cells but also on IgD⁺ B-cells. These results suggest that F-5-2 is a new type of B-cell mitogen.     

Human immunoglobulin class and IgG subclass regulation: Dual action of interleukin-4

Epstein-Barr virus (EBV) was used as a polyclonal human B cell mitogen to investigate the regulation of immunoglobulin class and IgG subclass responses by interleukin-4 (IL-4). Activation of tonsillar B cells with EBV resulted in an early peak of polyclonal immunoglobulin secretion between days 13 and 14 consisting of IgM, IgA, and IgG1, IgG2, IgG3 and IgG4, but not IgE. Addition of IL-4 to EBV-activated B cells at concentrations of 100 U/ml or greater induced the production of IgE and enhanced IgG4 secretion, but had no effect, or more often inhibited the other isotypes. In contrast, low concentrations of IL-4 (1-5 U/ml) significantly increased the production of IgM, IgA, IgG1, IgG2and IgG3, but had no effect on IgG4 or IgE. The increase in immunoglobulin secretion obtained with low concentrations of IL-4 was found to occur only with high-density (resting) B cells, suggesting that IL-4 was not functioning simply as a late-acting differentiation factor. Low concentrations of IL-4 significantly increased IgG1, IgG2, IgG3, and IgA production by surface (s)IgM⁺ (sIgG^?/sIgA^?) B cells which is consistent with heavy chain switching. In some experiments, however, IL-4 enhanced IgM secretion by sIgM⁺ B cells, and IgA, IgG1, IgG2, IgG3 by sIgM B cells, suggesting that it may have an additional B cell differentiation factor activity which was not isotype specific. The different effect of IL-4 at high and low concentrations were similar to those observed in B cell activation experiments, and may be due to the existence of high- and low-affinity IL-4 receptors.     

Circulating and pokeweed mitogen-induced immunoglobulin-secreting cells in systemic lupus erythematosus.

Peripheral blood mononuclear cells (PBM) obtained from patients with active untreated systemic lupus erythematosus (SLE) were evaluated both for the number of cells spontaneously secreting immunoglobulin (Ig) as well as for their capacity to generate immunoglobulin-secreting cells (ISC) in vitro in response to pokeweed mitogen (PWM). ISC were enumerated by a reverse haemolytic plaque assay designed to quantify the number of cells secreting IgG, IgM and IgA. PBM obtained from eight patients with active untreated SLE contained markedly increased numbers of ISC compared to age-, sex-, and race-matched normal control PBM. SLE PBM contained a mean of 13,805 +/- 3266 ISC per 10(6) cells, of which 74% secreted IgG, 10% IgM and 22% IgA, while normal PBM contained a mean of 779 +/- 143 ISC per 10(6) cells, with 57% secreting IgG, 25% IgM and 33% IgA. PBM obtained from SLE patients were also examined for their ability to generate ISC in vitro in response to PWM. SLE PBM were markedly deficient in their capacity to respond to PWM with the differentiation of ISC. This diminished responsiveness could not be ascribed to serum factors, the presence of increased numbers of cells with suppressive capacity or the absence of potentially responsive B cells. Rather, a deficiency of helper T cell activity appeared to be responsible. This was indicated by the observation that PWM responsiveness could be restored to SLE PBM by co-culturing them with purified mitomycin C-treated normal T cells.     

IgD class switching: Identification of a novel recombination site in neoplastic and normal B cells

IgD on normal B lymphocytes usually is co-expressed with IgM. A minority of normal plasma cells and rare B cell malignancies express exclusively IgD (IgM^?-IgD⁺). The low frequency has been explained by the lack of a recognizable switch region within the Cμ-Cδ intron. We analyzed four cases of IgM^?IgD⁺ hairy cell leukemia (HCL) by Southern (DNA) blot analysis and identified two cases with a recombinatorial event within the Cμ-Cδ intron and deletion of Cμ. DNA sequence analysis of junctional regions showed that Sμ or the immediate upstream region was used as a donor site and that the Cμ-Cδ intronic σδ region was used as acceptor site. Using polymerase chain reaction, we subsequently analyzed whether similar Sμ-σδ recombinations occur in normal tonsils containing IgM^?IgD⁺ plasma cells. Multiple products with a size range of 200–800 base pairs were detected in all four individuals, suggesting clustering of acceptor sites within σδ. Sequence analysis of three cloned products showed Sμ-σδ recombinations similar those observed in HCL. The σδ region contains a relatively high content of pentameric repeats with an extremely G-rich area and appears to function as a vestigial switch recombination site in normal and neoplastic IgM^?-IgD⁺ B cells.     

Identification of dendritic cells,B cell and T cell subsets in Tasmanian devil lymphoid tissue; evidence for poor immune cell infiltration into devil facial tumors

The Tasmanian devil is under threat of extinction due to the transmissible devil facial tumor disease (DFTD). This fatal tumor is an allograft that does not induce an immune response, raising questions about the activity of Tasmanian devil immune cells. T and B cell analysis has been limited by a lack of antibodies, hence the need to produce such reagents. Amino acid sequence analysis revealed that CD4, CD8, IgM, and IgG were closely related to other marsupials. Monoclonal antibodies were produced against CD4, CD8, IgM, and IgG by generating bacterial fusion proteins. These, and commercial antibodies against CD1a and CD83, identified T cells, B cells and dendritic cells by immunohistochemistry. CD4⁺ and CD8⁺ T cells were identified in pouch young thymus, adult lymph nodes, spleen, bronchus‐ and gut‐associated lymphoid tissue. Their anatomical distribution was characteristic of mammalian lymphoid tissues with more CD4⁺ than CD8⁺ cells in lymph nodes and splenic white pulp. IgM⁺ and IgG⁺ B cells were identified in adult lymph nodes, spleen, bronchus‐associated lymphoid tissue and gut‐associated lymphoid tissue, with more IgM⁺ than IgG⁺ cells. Dendritic cells were identified in lymph node, spleen and skin. This distribution is consistent with eutherian mammals and other marsupials, indicating they have the immune cell subsets for an anti‐tumor immunity. Devil facial tumor disease tumors contained more CD8⁺ than CD4⁺ cells, but in low numbers. There were also low numbers of CD1a⁺ and MHC class II⁺ cells, but no CD83⁺ IgM⁺ or IgG⁺ B cells, consistent with poor immune cell infiltration. Anat Rec, 297:925–938, 2014. © 2014 The Authors. The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology Published by Wiley Periodicals, Inc.     

Somatic mutation in autoantibody-associated VH genes of circulating IgM+IgD+ B cells

Naive B cells expressing IgM and IgD on their surface have no or little somatic mutations in V genes. We have demonstrated that the human IgM⁺IgD⁺B cell clone (0 – 81), which expresses nephritogenic idiotypes, produces IgM anti-DNA antibodies which show monospecificity to DNA. Using a DNA probe which specifically links to the V_H gene of antibody 0 – 81, we identified the counterpart germ-line V gene of 0 – 81, V3-7, which appears to be used by pathogenic autoantibodies in humans. Clone 0 – 81, which may belong to naive B cells in terms of cell phenotype, uses a somatically mutated V3-7 gene. We further studied DNA sequences of V3-7 genes in circulating IgM⁺IgD⁺B cells from normal subjects and patients with systemic lupus erythematosus (SLE). The results revealed that rearranged V3-7 genes in IgM⁺IgD⁺B cells from patients with SLE contained somatically mutated sequences at significantly increased frequencies. These data indicate an abnormal maturation of B cells in autoimmune states that may be associated with an escape of self-reactive B cells from the elimination process in the germinal center.     

Quantitation of Cells Secreting Immunoglobulins after Elution from Rheumatoid Synovial Tissue

Mononuclear cells (MNC) eluted from rheumatoid synovial tissue of 16 patients with rheumatoid arthritis were examined for immunoglobulin-secreting cells (ISC). Immediately after elution and separation synovial tissue MNC contained considerable numbers of ISC. IgG and IgA ISC were more abundant than IgM ISC. At the same time there were low numbers of ISC in blood. Synovial tissue ISC were lost during incubation with pokeweek mitogen (PWM), possibly because tissue MNC were already maximally triggered in vivo. This was in contrast to blood MNC, in which the number of ISC increased significantly during incubation with PWM.     

Phenotypic characteristics of thymic B lymphocytes in myasthenia gravis

We have found prominent secretion of immunoglobulin and anti-acetylcholine antibodies by thymic lymphocytes (TL) of myasthenics despite a relative paucity of B (surface IgM⁺) cells in such TL. To determine whether there was a surface IgM^– B cell in the TL which could manifest such responses, we compared the frequency of cells expressing the B cell-specific phenotypic marker CD20 (B1⁺), surface IgM (SIgM⁺), surface IgG (SIgG⁺), and surface IgD (SIgD⁺) in TL and autologous blood mononuclear cells in 36 myasthenic patients. B1⁺ cells were significantly more frequent than SIgM⁺ cells in TL (3.2±0.6 vs 0.6±0.2). In double-labeling studies, less than 25% of the B1⁺ cells coexpressed SIgM. Only 0.3% of the TL were SIgD⁺. In contrast, the frequencies of B1⁺ and SIgM⁺ cells in autologous blood were not significantly different (10.7±1.3 vs 8.2±0.8%). About 75% of blood B1⁺ cells co-expressed SIgM. These findings suggest that mast B cells in these TL have undergone isotope switching during priorin vivo differentiation and could manifest the observed humoral responses.     

Impact of the Yosemite Hantavirus Outbreak on Hantavirus Antibody Testing at a National Reference Laboratory

In conjunction with the 2012 Yosemite hantavirus outbreak, the number of sera our facility tested for hantavirus antibodies increased. We tracked test results and used the data set to determine if a more efficient testing algorithm was possible. Sera were screened using laboratory-developed pan-hantavirus IgG and IgM enzyme immunoassays (EIAs), with an index of >1.10 defined as positive. Sera that were IgM positive by screening (screen IgM⁺) were tested for Sin Nombre virus (SNV)-specific IgM using a laboratory-developed EIA; screen IgM⁺ IgG⁺ sera were also tested for SNV IgG using a laboratory-developed immunoblot assay. SNV antibody-positive samples were sent to state public health laboratories (PHL) or the CDC for confirmation. Of 3,946 sera tested from July through December 2012, 205 were screen IgM⁺ IgG negative (IgG⁻); 7/205 were SNV IgM⁺, but only 1/5 sent to PHL/CDC was confirmed as SNV IgM⁺. Of 61 screen IgM⁺ IgG⁺ sera, 16 were SNV antibody positive; 13/16 sera (from 11 patients) went to PHL/CDC, where SNV infection was confirmed for all patients. Of 12 confirmed patients, 7 had been exposed at Yosemite. A modified algorithm defining screen indices of ≥2.00 as positive identified 11/12 confirmed cases while reducing the number of sera requiring SNV-specific antibody testing by 65%; the patient missed was not tested until 3 months after the onset of symptoms. Hantavirus antibody testing at our facility identified 12 SNV-infected patients, including 7 exposed at Yosemite. Some screen IgM⁺ IgG⁻ SNV IgM⁺ results were false positives, emphasizing the value of PHL/CDC confirmatory testing. We identified a modified algorithm requiring analysis of fewer specimens for SNV-specific antibodies without loss of sensitivity.