- Email: [email protected]
Tolerant beekeepers display venom-specific functional IgG4 antibodies in the absence of specific IgE
Letters to the Editor Tolerant beekeepers display venom-specific functional IgG4 antibodies in the absence of specific IgE To the Editor: Beekeepers are exposed to multiple stings in season although they are immune to the effects apart from local swellings at the site of the sting. This is in contrast to venom-allergic individuals who are in general stung only occasionally yet remain at risk of IgE-mediated anaphylaxis. To assess the mechanism of tolerance in beekeepers, we measured their serum allergen-specific IgE and IgG4 levels. We measured the ability of their serum to form bee venom IgE-allergen complexes (the IgE-facilitated-allergen binding [FAB] assay). Results were compared to those in bee venom– allergic subjects (positive control) and normal healthy individuals (negative control). We also measured the ability of serum from tolerant beekeepers to inhibit allergen-IgE binding in the IgEFAB assay. To determine whether any observed differences in beekeepers are sustained in the absence of exposure, we compared immunoreactive and functional immunoglobulin levels in beekeepers inside/outside the beekeeping season. Approval was obtained from the local ethics committee, and the study was performed with the written informed consent of the participants. Sera from 10 nonallergic (tolerant) beekeepers (mean age, 48.2.years; men/women, 10/0) were analyzed in July, corresponding to the peak beekeeping season, and compared with sera from 10 bee venom–allergic subjects (mean age, 40.5.years; men/women, 4/6) and from 10 nonallergic healthy controls (mean age, 40.0 years; men/women, 4/6). Sera from beekeepers were additionally analyzed outside the season in January. Phospholipase A2-specific IgG4 and IgE levels were measured by using ImmunoCAP. IgE functional activity of participants’ sera was assessed by the IgE-FAB assay as described previously.1 Briefly, sera were incubated at 378C with 3 mg/mL of Apis mellifera extract. Allergen-IgE complexes were incubated for 1 hour at 48C to bind CD23 on the surface of B cells. Complexes bound to B cells were detected by flow cytometry by using a polyclonal human anti-IgE phycoerythrin-labeled antibody. In addition, serum inhibitory activity for IgE-FAB was measured in beekeepers’ serum obtained inside and outside the beekeeping season by incubating with an indicator serum (bee venom–allergic serum with elevated specific IgE level of >20 kU/mL) at 378C with 3 mg/mL of A mellifera extract. To explore the likely IgG association of this serum inhibitory activity, pooled serum from 3 bee-tolerant individuals was fractionated by affinity chromatography by using a Protein G column (Protein G minispin column, Generon, Berkshire, United Kingdom) and the inhibitory activity of the pooled serum was compared with that of IgGenriched and IgG-depleted fractions, as previously described.2 In tolerant beekeepers compared with bee venom–allergic individuals, there were marked elevations in allergen-specific IgG4 levels during the summer (median [interquartile range (IQR)], 76.00 [42.75-250.5]) (Fig 1, A) whereas allergen-specific IgE and IgE FAB were virtually absent (median [IQR], 0.14 [0.06-0.23]; 0.02 [0.0-0.80]) (Fig 1, B and C). In contrast, in venom-allergic subjects, there were markedly lower but detectable levels of specific IgG4 (median [IQR], 2.20 [0.91-10.55]) (Fig 1, A) whereas specific IgE and IgE FAB levels were elevated (median
[IQR], 4.84 [1.89-10.64]; 4.20 [3.59-5.74]) (Fig 1, B and C). In normal controls, there was complete absence of specific IgG4 (median [IQR], 0.05 [0.04-0.08]), IgE (median [IQR], 0.03 [0.020.08]), and IgE-FAB (median [IQR], 0.00 [0.0-0.27]) (Fig 1, A-C). To determine whether seasonal repeated field stings in tolerant bee keepers resulted in ‘‘protective’’ IgG-associated serum inhibitory activity (as observed during bee venom immunotherapy [VIT]), we measured the ability of their serum to inhibit IgEFAB by using an indicator system as described previously1 (Fig 2). Both IgG4 and serum inhibitory activity for IgE-FAB was markedly elevated in season (median [IQR], 76.00 [42.75250.5]; 75.63 [61.93-88.02]) (Fig 2, A and C). Although there was a small but significant decrease in both IgG4 and serum inhibitory activity out of season (median [IQR], 47.50 [10-188]; 71.04 [43.34-77.10]) (Fig 2, A and C), levels remained substantially elevated, whereas serum IgE was virtually undetectable both inside and outside of season (Fig 2, B). Separation of pooled sera into IgG-containing and IgG-depleted fractions confirmed that greater than 90% of the serum inhibitory activity for bee venom allergen-IgE binding to B cells resided within the IgG fraction (data not shown). Our findings in beekeepers have similarities to those observed during bee VIT where significant increases in bee venom–specific IgG4 serum antibody levels during VIT maintenance3 were observed, likely due to an accompanying increase in IL-10–secreting regulatory T cells.4 In our studies of bee VIT in children, we also found parallel significant increases in serum inhibitory activity for IgE-FAB.3 However, high levels of allergen-specific IgG4 antibodies and serum inhibitory activity for IgE-FAB were not sustained at 2 years following VIT withdrawal. In tolerant beekeepers, venom-specific IgG4 levels remained significantly elevated between seasons as compared with both normal controls and venom-allergic patients with a small but significant decline during winter in the absence of stings. In our previous study of bee VIT,3 we observed a significant decline in venom-specific serum IgE antibodies during VIT with a further decline at 2 years following VIT withdrawal. We speculated that long-term clinical protection following bee VIT withdrawal5 may result from the prolonged suppression of allergen-specific IgE rather than sustained elevations in blocking IgG4 antibodies. Similarly, we identified low/absent levels of specific IgE in our beekeepers. Our current data in tolerant beekeepers suggest that their protection from allergic reactions afforded by repeated stings may be due to either high levels of immunoreactive IgG4 antibodies with blocking activity and/or low levels of venom-specific IgE antibodies. With regard to the underlying mechanism of long-term tolerance in beekeepers, this dichotomy could be further addressed in a future study involving long-term withdrawal from beekeeping, although this may be difficult to achieve for cultural reasons. Our findings in tolerant beekeepers of a decline in immunoreactive IgG4 antibody levels between seasons, and in our previous study of VIT in children, are in contrast to studies of antibody levels during grass pollen immunotherapy.6 Grass pollen–treated individuals maintained persistent IgG-associated serum inhibitory activity for FAB for at least 2 years following the withdrawal of immunotherapy. One explanation for differences in protection afforded by beekeeping or bee VIT compared with grass pollen 1419
1420 LETTERS TO THE EDITOR
J ALLERGY CLIN IMMUNOL MAY 2013
FIG 1. A, Serum PLA2-specific IgG4 antibodies. B, Serum PLA2-specific IgE antibodies. C, The level of serum IgE–associated binding activity for allergen-IgE complexes to B cells. Data are given for tolerant beekeepers (BK), bee venom–allergic controls (AC), and nonatopic normal healthy controls (NC). Mann-Whitney test (P < .05 considered significant). PLA2, Phospholipase A2.
P =.02
300
200
100
0
C
IgE
25.0
PLA2 -specific IgE (kU/L)
400
PLA2 -specific IgG4 (mgA/L)
B
IgG4
P = ns
20.0 15.0 10.0 5.0
Out
P = . 002
90
60
30
0
0.0
In
Inhibition of IgE-FAB
120
Inhibition of IgE-FAB (%)
A
In
Out
In
Out
BK FIG 2. A, Serum PLA2-specific IgG4 antibodies. B, Serum PLA2-specific IgE antibodies. C, The level of serum IgG–associated inhibitory activity for binding of allergen-IgE complexes to B cells. Data are given for tolerant beekeepers inside (July) and outside (January) the beekeeping season. Wilcoxon matched-pairs signed ranks test (P < .05 considered significant). PLA2, Phospholipase A2.
immunotherapy may be the intermittent and systemic exposure to venom that contrasts with the low-dose, particulate and mucosal exposure to grass pollen that is responsible for the disease and alternative mechanisms of tolerance induction. Eva-Maria Varga, MDa Fahima Kausar, MScb Werner Aberer, MDc Maximilian Zach, MDa Ernst Eber, MDa Stephen R. Durham, MDb Mohamed H. Shamji, PhDb From athe Respiratory and Allergic Disease Division, Department of Paediatrics, Medical University Graz, Austria; bAllergy and Clinical Immunology, National Heart and
Lung Institute, Imperial College London, United Kingdom; and cthe Department of Environmental Dermatology and Allergy, Medical University Graz, Austria. E-mail: [email protected]. Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.
REFERENCES 1. Shamji MH, Wilcock LK, Wachholz PA, Dearman RJ, Kimber I, Wurtzen PA, et al. The IgE-facililated allergen binding (FAB) assay: validation of a novel flowcytometric based method for the detection of inhibitory antibody responses. J Immunol Methods 2006;317:71-9. 2. Wachholz PA, Soni NK, Till SJ, Durham SR. Inhibition of allergen-IgE binding to B cells by IgG antibodies after grass pollen immunotherapy. J Allergy Clin Immunol 2003;112:915-22.
LETTERS TO THE EDITOR 1421
J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 5
3. Varga EM, Francis JN, Zach MS, Klunker S, Aberer W, Durham SR. Time course of serum inhibitory activity for facilitated allergen-IgE binding during bee venom immunotherapy in children. Clin Exp Allergy 2009;39:1353-7. 4. Meiler F, Zumkehr J, Klunker S, R€uckert B, Akdis CA, Akdis M. In vivo switch to IL-10-secreting T regulatory cells in high dose allergen exposure. J Exp Med 2008; 205:2887-98. 5. Golden DBK, Kagey-Sobotka A, Norman PS, Hamilton RG, Lichtenstein L. Outcomes of allergy to insect stings in children with and without venom immunotherapy. N Engl J Med 2004;351:668-74. 6. James LK, Shamji MH, Walker SM, Wilson DR, Wachholz PA, Francis JN, et al. Long-term tolerance after allergen immunotherapy is accompanied by selective persistence of blocking antibodies. J Allergy Clin Immunol 2011;127:509-16. Available online October 11, 2012. http://dx.doi.org/10.1016/j.jaci.2012.08.037
Adult-onset manifestation of idiopathic T-cell lymphopenia due to a heterozygous RAG1 mutation To the Editor: We would like to share a relevant and interesting immunodeficiency case that would be of interest to the readers of the Journal of Allergy and Clinical Immunology. This report describes an adult-onset idiopathic T-cell lymphopenia due to recombinase activating gene 1 (RAG1) deficiency in an HIV-negative male patient with no recurrent or opportunistic infections presenting at the age of 38 years with chronic dermatitis, pruritus, and hyperkeratosis. Clinical and immunologic examination revealed eosinophilia with modestly elevated IgE (747 kU/L), normal IgG, IgA, and IgM, profound pan–T-cell lymphopenia, high normal total B cells, and slightly reduced natural killer cells. Genetic analysis revealed a heterozygous frameshift mutation in the RAG1 gene, resulting in a truncated RAG1 protein. This is a late clinical presentation of an idiopathic T-cell lymphopenia secondary to a heterozygous hypomorphic RAG1 mutation and has important implications for the considerable phenotypic variability related to molecular defects in genes typically associated with severe combined immunodeficiency or Omenn syndrome (OS). A 38-year-old man presented with significant pruritic skin rash on his legs and eosinophilia that had been present for 2 years with poor resolution using over-the-counter topical treatments. Medical history revealed a healthy childhood and adulthood until 2 years earlier when the skin rash first appeared on the lower extremities. Gross examination of the skin revealed a generalized dermatitis with hyperkeratosis without histologic evidence of granulomas (Fig 1, A). There was also keratoderma of the feet and onychodystrophy. Skin biopsy of the lesions on the leg revealed a chronic dermatitis with eosinophilia, which was negative for IgG, IgM, or IgA with weak, discontinuous granular deposition of C3 along the basement membrane zone. Complete blood cell count with differential revealed significant lymphopenia, which on detailed lymphocyte subset evaluation by flow cytometry revealed profound pan–T-cell lymphopenia (Table I). There was a relative increase in the frequency of activated T cells (CD41CD251 and HLA-DR1 CD4 and CD8 T cells). Thymic function was significantly impaired for age, with almost absent CD4 recent thymic emigrants and TREC. Genetic analysis revealed the following heterozygous frameshift mutation (c.256_257delAA, K86VfsX33) in exon 2 of the gene encoding RAG1 that has been previously reported to be associated with SCID and OS, when present as a homozygous or compound heterozygous mutation.1 No other pathogenic genetic variations were found in either
the known coding or promoter regions of the RAG1 gene or in any of the following SCID-associated genes: RAG2, JAK3, IL7R, ADA, CD3D, CD3E, DCLRE1C, and IL2RG. Genetic analysis for the above RAG1 mutation was performed not only on DNA extracted from whole blood but also from PBMCs and buccal brushing. In all 3 sample types, the same heterozygous mutation was identified, indicating that this was not a somatic mutation but a germline variation in the RAG1 gene. Genetic testing for this specific RAG1 mutation was performed on PBMCs isolated from whole blood of both parents of this patient, and the father was shown to have the same heterozygous mutation as the patient; however, the father was clinically, phenotypically, and immunologically normal with normal T, B, and natural killer cell counts. A second mutation was not identified in the patient or the mother. The patient has a single older male sibling who was not evaluated but was negative for relevant clinical history. Further immunologic analysis revealed robust antibody responses to vaccine antigens such as tetanus and diphtheria toxoids, but significantly abnormal lymphocyte proliferation to mitogens (PHA, PWM) and antigens (Candida and tetanus toxoid) and stimulation with soluble anti-CD3. RAG1 function was determined by recombinase activity (Fig 1, B), and the 256-257delAA mutant had only 2.67% 6 0.58% activity as compared with wild-type RAG1. Although overexpression experiments have shown that this mutation can induce the production of an N-terminal–truncated protein by using a second internal ATG1 (which might explain the residual recombination activity), we failed to detect the expression of the mutant RAG1 on retrovirus-mediated transduction of Rag12/2 pro–B cells. Detailed B-cell subset analysis revealed low total memory B cells with low class-switched memory and marginal zone B cells with a significant increase in naive B cells (CD272) and transitional B cells (CD191CD2411CD3811). Functional B-cell analysis revealed that the patient’s B cells were capable of plasmablast differentiation on stimulation with CD40L and IL-21 or CpG, though it was decreased than that in a healthy control (data not shown). B-cell proliferation in response to CD40L 1 IL-21 was lower than in control B cells, but almost comparable to that in control with CpG. Also, the distribution of immunoglobulin light chains (kappa/lambda) was normal, arguing against significant defects of receptor editing of B cells. Therefore, it would appear that patient’s B cells are capable of responding to helper T-cell signals, and the reduced frequency of memory B cells is likely related to the low numbers of such T cells. There was evidence of autoantibody production with high titer of ANA, doublestranded DNA and SS-A antigens in addition to antibodies to 14 other autoantigens. Although there was no clinical evidence of specific autoimmune disease or granulomatous disease and renal function was preserved,2,3 the diffuse skin rash, which was responsive to steroids, suggests that a clear inflammatory process was present (prior to treatment). Molecular T-cell receptor repertoire analysis (T-cell receptor Vb spectratyping) revealed an overall polyclonal T-cell repertoire for most T-cell receptor Vb families with oligoclonality of a few (family 2 was absent and families 3-1, 6-4, 13, and 16 were oligoclonal with limited diversity). There was a significant increase in peripheral levels of IL-7, which is related to the presence of T-cell lymphopenia. Clinically, the patient responded well to wet dressings with ammonium lactate topical (AmLactin), a keratolytic, and treatment with triamcinolone. He was treated with prophylactic antibiotics with no evidence of infection.
[IQR], 4.84 [1.89-10.64]; 4.20 [3.59-5.74]) (Fig 1, B and C). In normal controls, there was complete absence of specific IgG4 (median [IQR], 0.05 [0.04-0.08]), IgE (median [IQR], 0.03 [0.020.08]), and IgE-FAB (median [IQR], 0.00 [0.0-0.27]) (Fig 1, A-C). To determine whether seasonal repeated field stings in tolerant bee keepers resulted in ‘‘protective’’ IgG-associated serum inhibitory activity (as observed during bee venom immunotherapy [VIT]), we measured the ability of their serum to inhibit IgEFAB by using an indicator system as described previously1 (Fig 2). Both IgG4 and serum inhibitory activity for IgE-FAB was markedly elevated in season (median [IQR], 76.00 [42.75250.5]; 75.63 [61.93-88.02]) (Fig 2, A and C). Although there was a small but significant decrease in both IgG4 and serum inhibitory activity out of season (median [IQR], 47.50 [10-188]; 71.04 [43.34-77.10]) (Fig 2, A and C), levels remained substantially elevated, whereas serum IgE was virtually undetectable both inside and outside of season (Fig 2, B). Separation of pooled sera into IgG-containing and IgG-depleted fractions confirmed that greater than 90% of the serum inhibitory activity for bee venom allergen-IgE binding to B cells resided within the IgG fraction (data not shown). Our findings in beekeepers have similarities to those observed during bee VIT where significant increases in bee venom–specific IgG4 serum antibody levels during VIT maintenance3 were observed, likely due to an accompanying increase in IL-10–secreting regulatory T cells.4 In our studies of bee VIT in children, we also found parallel significant increases in serum inhibitory activity for IgE-FAB.3 However, high levels of allergen-specific IgG4 antibodies and serum inhibitory activity for IgE-FAB were not sustained at 2 years following VIT withdrawal. In tolerant beekeepers, venom-specific IgG4 levels remained significantly elevated between seasons as compared with both normal controls and venom-allergic patients with a small but significant decline during winter in the absence of stings. In our previous study of bee VIT,3 we observed a significant decline in venom-specific serum IgE antibodies during VIT with a further decline at 2 years following VIT withdrawal. We speculated that long-term clinical protection following bee VIT withdrawal5 may result from the prolonged suppression of allergen-specific IgE rather than sustained elevations in blocking IgG4 antibodies. Similarly, we identified low/absent levels of specific IgE in our beekeepers. Our current data in tolerant beekeepers suggest that their protection from allergic reactions afforded by repeated stings may be due to either high levels of immunoreactive IgG4 antibodies with blocking activity and/or low levels of venom-specific IgE antibodies. With regard to the underlying mechanism of long-term tolerance in beekeepers, this dichotomy could be further addressed in a future study involving long-term withdrawal from beekeeping, although this may be difficult to achieve for cultural reasons. Our findings in tolerant beekeepers of a decline in immunoreactive IgG4 antibody levels between seasons, and in our previous study of VIT in children, are in contrast to studies of antibody levels during grass pollen immunotherapy.6 Grass pollen–treated individuals maintained persistent IgG-associated serum inhibitory activity for FAB for at least 2 years following the withdrawal of immunotherapy. One explanation for differences in protection afforded by beekeeping or bee VIT compared with grass pollen 1419
1420 LETTERS TO THE EDITOR
J ALLERGY CLIN IMMUNOL MAY 2013
FIG 1. A, Serum PLA2-specific IgG4 antibodies. B, Serum PLA2-specific IgE antibodies. C, The level of serum IgE–associated binding activity for allergen-IgE complexes to B cells. Data are given for tolerant beekeepers (BK), bee venom–allergic controls (AC), and nonatopic normal healthy controls (NC). Mann-Whitney test (P < .05 considered significant). PLA2, Phospholipase A2.
P =.02
300
200
100
0
C
IgE
25.0
PLA2 -specific IgE (kU/L)
400
PLA2 -specific IgG4 (mgA/L)
B
IgG4
P = ns
20.0 15.0 10.0 5.0
Out
P = . 002
90
60
30
0
0.0
In
Inhibition of IgE-FAB
120
Inhibition of IgE-FAB (%)
A
In
Out
In
Out
BK FIG 2. A, Serum PLA2-specific IgG4 antibodies. B, Serum PLA2-specific IgE antibodies. C, The level of serum IgG–associated inhibitory activity for binding of allergen-IgE complexes to B cells. Data are given for tolerant beekeepers inside (July) and outside (January) the beekeeping season. Wilcoxon matched-pairs signed ranks test (P < .05 considered significant). PLA2, Phospholipase A2.
immunotherapy may be the intermittent and systemic exposure to venom that contrasts with the low-dose, particulate and mucosal exposure to grass pollen that is responsible for the disease and alternative mechanisms of tolerance induction. Eva-Maria Varga, MDa Fahima Kausar, MScb Werner Aberer, MDc Maximilian Zach, MDa Ernst Eber, MDa Stephen R. Durham, MDb Mohamed H. Shamji, PhDb From athe Respiratory and Allergic Disease Division, Department of Paediatrics, Medical University Graz, Austria; bAllergy and Clinical Immunology, National Heart and
Lung Institute, Imperial College London, United Kingdom; and cthe Department of Environmental Dermatology and Allergy, Medical University Graz, Austria. E-mail: [email protected]. Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.
REFERENCES 1. Shamji MH, Wilcock LK, Wachholz PA, Dearman RJ, Kimber I, Wurtzen PA, et al. The IgE-facililated allergen binding (FAB) assay: validation of a novel flowcytometric based method for the detection of inhibitory antibody responses. J Immunol Methods 2006;317:71-9. 2. Wachholz PA, Soni NK, Till SJ, Durham SR. Inhibition of allergen-IgE binding to B cells by IgG antibodies after grass pollen immunotherapy. J Allergy Clin Immunol 2003;112:915-22.
LETTERS TO THE EDITOR 1421
J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 5
3. Varga EM, Francis JN, Zach MS, Klunker S, Aberer W, Durham SR. Time course of serum inhibitory activity for facilitated allergen-IgE binding during bee venom immunotherapy in children. Clin Exp Allergy 2009;39:1353-7. 4. Meiler F, Zumkehr J, Klunker S, R€uckert B, Akdis CA, Akdis M. In vivo switch to IL-10-secreting T regulatory cells in high dose allergen exposure. J Exp Med 2008; 205:2887-98. 5. Golden DBK, Kagey-Sobotka A, Norman PS, Hamilton RG, Lichtenstein L. Outcomes of allergy to insect stings in children with and without venom immunotherapy. N Engl J Med 2004;351:668-74. 6. James LK, Shamji MH, Walker SM, Wilson DR, Wachholz PA, Francis JN, et al. Long-term tolerance after allergen immunotherapy is accompanied by selective persistence of blocking antibodies. J Allergy Clin Immunol 2011;127:509-16. Available online October 11, 2012. http://dx.doi.org/10.1016/j.jaci.2012.08.037
Adult-onset manifestation of idiopathic T-cell lymphopenia due to a heterozygous RAG1 mutation To the Editor: We would like to share a relevant and interesting immunodeficiency case that would be of interest to the readers of the Journal of Allergy and Clinical Immunology. This report describes an adult-onset idiopathic T-cell lymphopenia due to recombinase activating gene 1 (RAG1) deficiency in an HIV-negative male patient with no recurrent or opportunistic infections presenting at the age of 38 years with chronic dermatitis, pruritus, and hyperkeratosis. Clinical and immunologic examination revealed eosinophilia with modestly elevated IgE (747 kU/L), normal IgG, IgA, and IgM, profound pan–T-cell lymphopenia, high normal total B cells, and slightly reduced natural killer cells. Genetic analysis revealed a heterozygous frameshift mutation in the RAG1 gene, resulting in a truncated RAG1 protein. This is a late clinical presentation of an idiopathic T-cell lymphopenia secondary to a heterozygous hypomorphic RAG1 mutation and has important implications for the considerable phenotypic variability related to molecular defects in genes typically associated with severe combined immunodeficiency or Omenn syndrome (OS). A 38-year-old man presented with significant pruritic skin rash on his legs and eosinophilia that had been present for 2 years with poor resolution using over-the-counter topical treatments. Medical history revealed a healthy childhood and adulthood until 2 years earlier when the skin rash first appeared on the lower extremities. Gross examination of the skin revealed a generalized dermatitis with hyperkeratosis without histologic evidence of granulomas (Fig 1, A). There was also keratoderma of the feet and onychodystrophy. Skin biopsy of the lesions on the leg revealed a chronic dermatitis with eosinophilia, which was negative for IgG, IgM, or IgA with weak, discontinuous granular deposition of C3 along the basement membrane zone. Complete blood cell count with differential revealed significant lymphopenia, which on detailed lymphocyte subset evaluation by flow cytometry revealed profound pan–T-cell lymphopenia (Table I). There was a relative increase in the frequency of activated T cells (CD41CD251 and HLA-DR1 CD4 and CD8 T cells). Thymic function was significantly impaired for age, with almost absent CD4 recent thymic emigrants and TREC. Genetic analysis revealed the following heterozygous frameshift mutation (c.256_257delAA, K86VfsX33) in exon 2 of the gene encoding RAG1 that has been previously reported to be associated with SCID and OS, when present as a homozygous or compound heterozygous mutation.1 No other pathogenic genetic variations were found in either
the known coding or promoter regions of the RAG1 gene or in any of the following SCID-associated genes: RAG2, JAK3, IL7R, ADA, CD3D, CD3E, DCLRE1C, and IL2RG. Genetic analysis for the above RAG1 mutation was performed not only on DNA extracted from whole blood but also from PBMCs and buccal brushing. In all 3 sample types, the same heterozygous mutation was identified, indicating that this was not a somatic mutation but a germline variation in the RAG1 gene. Genetic testing for this specific RAG1 mutation was performed on PBMCs isolated from whole blood of both parents of this patient, and the father was shown to have the same heterozygous mutation as the patient; however, the father was clinically, phenotypically, and immunologically normal with normal T, B, and natural killer cell counts. A second mutation was not identified in the patient or the mother. The patient has a single older male sibling who was not evaluated but was negative for relevant clinical history. Further immunologic analysis revealed robust antibody responses to vaccine antigens such as tetanus and diphtheria toxoids, but significantly abnormal lymphocyte proliferation to mitogens (PHA, PWM) and antigens (Candida and tetanus toxoid) and stimulation with soluble anti-CD3. RAG1 function was determined by recombinase activity (Fig 1, B), and the 256-257delAA mutant had only 2.67% 6 0.58% activity as compared with wild-type RAG1. Although overexpression experiments have shown that this mutation can induce the production of an N-terminal–truncated protein by using a second internal ATG1 (which might explain the residual recombination activity), we failed to detect the expression of the mutant RAG1 on retrovirus-mediated transduction of Rag12/2 pro–B cells. Detailed B-cell subset analysis revealed low total memory B cells with low class-switched memory and marginal zone B cells with a significant increase in naive B cells (CD272) and transitional B cells (CD191CD2411CD3811). Functional B-cell analysis revealed that the patient’s B cells were capable of plasmablast differentiation on stimulation with CD40L and IL-21 or CpG, though it was decreased than that in a healthy control (data not shown). B-cell proliferation in response to CD40L 1 IL-21 was lower than in control B cells, but almost comparable to that in control with CpG. Also, the distribution of immunoglobulin light chains (kappa/lambda) was normal, arguing against significant defects of receptor editing of B cells. Therefore, it would appear that patient’s B cells are capable of responding to helper T-cell signals, and the reduced frequency of memory B cells is likely related to the low numbers of such T cells. There was evidence of autoantibody production with high titer of ANA, doublestranded DNA and SS-A antigens in addition to antibodies to 14 other autoantigens. Although there was no clinical evidence of specific autoimmune disease or granulomatous disease and renal function was preserved,2,3 the diffuse skin rash, which was responsive to steroids, suggests that a clear inflammatory process was present (prior to treatment). Molecular T-cell receptor repertoire analysis (T-cell receptor Vb spectratyping) revealed an overall polyclonal T-cell repertoire for most T-cell receptor Vb families with oligoclonality of a few (family 2 was absent and families 3-1, 6-4, 13, and 16 were oligoclonal with limited diversity). There was a significant increase in peripheral levels of IL-7, which is related to the presence of T-cell lymphopenia. Clinically, the patient responded well to wet dressings with ammonium lactate topical (AmLactin), a keratolytic, and treatment with triamcinolone. He was treated with prophylactic antibiotics with no evidence of infection.