Cryofibrinogenemia triggered by a monoclonal paraprotein successfully treated with cyclophosphamide.

Cryofibrinogenemia is a rare clinical finding with a yet unknown physiopathogenic mechanism. We describe the case of a woman with cold-induced extensive necrotic lesions that responded poorly to initial corticosteroid and anticoagulant therapies. Serum cryoglobulin determinations were persistently negative. After several years of evolution, she developed severe cold-related skin lesions that required left-leg amputation. Further analysis disclosed the presence of cryofibrinogen and an apparently insignificant serum monoclonal immunoglobulin Gκ peak. We additionally demonstrate that the cold precipitation of fibrinogen was directly related to the monoclonal paraprotein presence. The lesions responded dramatically to a B cell-targeted therapy with intravenous cyclophosphamide and dexamethasone.

A review of hypersensitivity methods to detect immune responses to SARS-CoV-2.

The investigation of the immune response after SARS-CoV-2 infection has been the goal of many researchers worldwide. The study of humoral immune responses and in vitro T cell production after infection requires the obtaining of individualized blood samples to test the presence of antibodies or activated T cells specific for the virus. In vitro T cell studies are especially troublesome due to the need for more specialized resources often outside the daily routine of clinical laboratories. For this reason the development of a simple and objective method to achieve these T cell studies is needed. In this manuscript we reviewed the hypersensitivity reactions, the theoretical basis and the historical background of delayed type hypersensitivity (DTH) which uses the principles of use of this test in the clinical setting for the past century. In the second part of the review, we focus on COVID adaptive immune responses, to understand the differences and challenges offered by this new application of DTH to investigate immune responses elicited after infection. In the last part of the review a vision provided for the use of this test to investigate the immunogenicity elicited by the vaccines. In our opinion, the clinical guidelines of immune assessment of SARS-CoV-2-infected or vaccinated individuals should include this simple and low-cost test to measure T-cell immunity. Rationale and improved vaccination schemes could be obtained after its implementation in the routine assessment of immunity in this pandemic situation.

Rearrangement of only one human IGHV gene is sufficient to generate a wide repertoire of antigen specific antibody responses in transgenic mice.

In recent years, mice carrying human IG transgenes are being generated for the production of human monoclonal antibodies as an alternative approach to the conventional use of mouse or chimeric-humanized antibodies. Theoretically, the size of the repertoire of human antibodies that these mice could produce would be critically dependent on the number of human V genes introduced in the transgene. This could be the case for BABkappa and BABkappa,lambda transgenic mice, which carry several genes from the human IGK (BABkappa), and IGK and IGL (BABkappa,lambda) loci, but only five human IGHV genes and the entire IGHD-IGHJ cluster linked to two human IGHC (IGHM-IGHD) genes. We analyzed the expressed human IG genes in 30 IgM-secreting hybridomas generated from transgenic mice immunized either with soluble proteins (human IgM coupled to KLH) or with cells (human PBMC, tumour cell lines or rat cells transfected with human CD69). The results show that all hybridoma cells analyzed rearranged exclusively the IGHV1-2 gene, in contrast with naive spleen B cells that used three out of the five IGHV genes present in the transgene. The configuration of the rearranged CDR3 region revealed a much higher heterogeneity in the heavy chains. A variety of IGHJ and IGHD genes were used in hybridomas, and somatic mutations were also seen in some hybrids. Regarding the rearranged light chains genes, it was a much higher variety in the use of V and J genes in both, kappa and lambda chains, than in the heavy chain, and also in the level of mutation. The results indicate that only one IGHV gene is sufficient to generate a wide repertoire of antigen specific antibody responses. Thus, efforts aimed at the generation of new transgenic mice should focus more on the integrity of the D/J region and on the DNA regions regulating somatic hypermutation, rather than on the number of V genes present in the transgene.

Human monoclonal antibodies produced in transgenic BABkappa,lambda mice recognising idiotypic immunoglobulins of human lymphoma cells.

Clonal idiotypic immunoglobulins of follicular lymphomas can be isolated by somatic fusion procedures. Idiotypic IgMs (Id-IgM) were isolated from two patients and used to immunise a strain of mice, deficient in mouse antibody production and engineered with yeast artificial chromosomes (YAC) containing fragments of the human immunoglobulin (Ig) micro/delta heavy chain and kappa/lambda light chain loci. Sequence analysis showed that hybridomas prepared from spleen cells of immunised mice expressed exclusively one of the six VH genes (VH1-2) present in the YAC transgene with different D/J rearrangements, and secrete fully human monoclonal antibodies (mAb) that recognised the tumour-specific IgM proteins. Further studies of the reactivity of the monoclonal anti-human Id-IgM antibodies revealed that they are specific for the individual protein of each patient and probably react with idiotypic determinants. In one case studied, the antibody recognised specifically the lymphoma cell expressing the corresponding idiotypic IgM and lysed those cells in the presence of complement. This is the first example of a human monoclonal antibody with such characteristics and may be of further use in the therapy of patients with B cell malignancies.

Anti-idiotypic Immunotherapy in follicular lymphoma patients: results of a long follow-up study.

A specific immune response against the individual idiotypic determinants of clonal immunoglobulins can be elicited in patients with B-cell malignancies. We analyze the clinical outcome and the presence of tumor cells in the blood of 8 patients with follicular lymphoma, vaccinated with autologous idiotype protein from their lymphoma cells. After a median follow-up of 90 months (range: 54 to 128), all patients, except 1, remain in complete clinical remission and 5 are in complete molecular remission. Our results suggest that idiotype vaccination induces long-lasting clinical and molecular remissions and constitutes a potential curative treatment in follicular lymphoma patients.

Intraclonal variability of VH genes in follicular lymphoma patients who have received anti-idiotypic immunotherapy.

Subclonal heterogeneity can affect idiotypic determinants present in the clonotypic immunoglobulin of B-cell follicular lymphomas (FLs) and may limit the effect of antilymphoma treatments performed by immunization of patients with their own tumor-associated idiotypic immunoglobulin. Idiotype-secreting hybridomas were obtained by fusion of tumor cells from 5 patients with FL, and the K6H6/B5 human heteromyeloma and rearranged VH genes from tumor samples and hybridomas were amplified, cloned, and sequenced. Sequences were aligned with germline genes and somatic mutations, intraclonal heterogeneity and genealogic relations of the B-cell clones in the different biopsy specimens were determined. The VH sequence of the progenitor clone was determined in samples of the tumoral population. Further diversification resulted in the presence of 2 to 6 subclones in 4 of the 5 samples studied. Only in 1 patient did the hypermutation mechanism introduce differences among most of the potential idiotopes present in individual subclones. The VH sequence of the hybridoma that provided the idiotypic-vaccine was identified in one of the tumor subclones in all cases. No relapse has been demonstrated in 3 of the 4 vaccinated patients (follow-up: 29-103 months). We conclude that despite potential differences in the idiotypic region expressed by individual tumor cells, at least some potential idiotopes may be preserved among all the tumor subclones in most cases studied. All vaccinated patients developed immune responses against the autologous tumor idiotypic immunoglobulin. Polyclonal anti-idiotypic immune responses induced with a vaccine obtained from 1 hybridoma may be effective against all the idiotypic variants present in the tumor population.

Anti-idiotypic vaccination in the treatment of low-grade B-cell lymphoma.

BACKGROUND AND OBJECTIVES: Patients with B-cell lymphoma can be induced to mount a specific immune response against the individual idiotypic determinants expressed in their tumor cells. This form of active immunotherapy is now under evaluation in the clinical setting. We evaluated the feasibility and effectiveness of this kind of immunotherapy in a group of patients with low-grade lymphoma, which included two cases of bi/triclonal lymphoma. DESIGN AND METHODS: Nine patients with a histopathologic diagnosis of follicular non-Hodgkin's (NHL) low-grade B-cell lymphoma were initially selected for this disease-free survival study. Idiotypic proteins were recovered by somatic fusion of the tumor cells and their identity with the tumor idiotype determined by molecular methods. The patients received the vaccine consisting of their tumor Ig protein coupled to keyhole limpet hemocyanine and were observed for toxicity, anti-idiotypic immune response, clinical outcome and circulating t(14;18)+ tumor cells. RESULTS: The median duration of follow-up was 40 (10-64) months from the initiation of immunotherapy. Tumor regression was detected in two patients. No tumor progression was observed in the other patients. Eight patients generated specific anti-idiotypic antibodies and 3 out of five were cleared of circulating t(14;18)+ cells. INTERPRETATION AND CONCLUSIONS: Induction of tumor-specific anti-idiotypic immune responses may be of benefit to patients affected by low-grade B-cell NHL. Our results are in line with those previously reported and call attention to the issue of tumor clonality in this kind of treatment.