Here, we present the medical history of a patient with treatment refractory ITP and persistent lymphadenopathy diagnosed at the age of 11-years, who was splenectomized and received multiple courses of high-dose corticosteroid before he was eventually treated with Rituximab resulting in a sustained response with normalization of thrombocyte counts. However, in the setting of severe pneumococcal meningitis, hypogammaglobulinemia was diagnosed, and an extensive immunological investigation was performed in order to characterize the immune status of the patient, and to distinguish between a primary immunodeficiency and a side effect of Rituximab treatment.
Common variable immunodeficiency
The hallmark of CVID is increased susceptibility to bacterial respiratory tract infections caused by reduced levels of serum immunoglobulins. CVID is defined as significantly reduced levels of IgG and decreased levels of IgA and/or IgM together with an absence of iso-haemagglutinins and/or inappropriate responses to pneumococcal vaccination [1, 2]. Several different classification systems have been developed in order to divide this broad spectrum of immunodeficiencies into biologically and clinically meaningful groups predicting severity of disease, treatment response, and prognosis [7, 8]. The Freiburg classification allows a division of patients into three distinct groups and emphasizes that the central characteristic of CVID is lack of switched memory B cells (IgD- IgM- CD27+) [8, 9], which is associated with the development of autoimmunity, lymphoid hyperplasia, splenomegaly, and granulomatous disease [10, 11] and therefore a major determinant of disease severity. Another aspect of CVID is that somatic hypermutation is impaired in B cells of about 70% of patients, thus interfering with the production of highly specific antibodies . Although CVID primarily affects the B cell population and immunoglobulin synthesis, it may also influence T cell immunity. Numerous abnormalities in the T cell compartment have been reported in CVID patients, including T lymphopenia, decreased CD4 + CD45RA + T cells, a restricted T cell repertoire, accelerated T cell death, and impaired cytokine production . As to the origin of CVID, a number of genetic defects, including mutations in TACI, ICOS, BAFF-R, CD19, CD20, and CD81, which are all molecules involved in differentiation, survival, and activation of B cells, have been identified. These only account for a minority of CVID cases (about 15-20%), and the molecular pathophysiology of the remaining fraction has remained largely obscure . Recently, however, a genome-wide association study has added novel mutations to this list by identifying diverse causes of CVID . Taken together, CVID cannot simply be characterized as a condition of impaired immune responses, but should rather be viewed as state of aberrant immunity.
Autoimmunity in CVID
A substantial proportion of patients with CVID also experience autoimmune manifestations, ranging from 22% to 48% in different studies, which is associated with the presence of granulomatous disease and TACI mutations [3, 7]. Among autoimmune conditions associated with CVID, cytopenias are particularly common, often present together with splenomegaly . In different case series, ITP has been diagnosed in 7% to 20% of patients with CVID [7, 16, 17]. In the context of the present case it is notable that the diagnosis of ITP and/or AIHA frequently precedes that of CVID, which was indeed the case in 54% of patients in one study  and in 62% in another study . Besides ITP and AIHA, other hematologic manifestations overrepresented among CVID patients are polyclonal lymphocytic infiltration and lymphoid malignancy, each of which is positively correlated with the serum IgM level . Several hypotheses have been presented to explain the increased frequency of autoimmune manifestations in CVID. Given that both defective B cell class-switch and loss of somatic hypermutation are correlated to autoimmunity it has been suggested that such molecular alterations may explain the inability to exclude autoreactive B cell clones .
Clinical use of Rituximab
Rituximab is an anti-CD20 humanized chimeric monoclonal antibody and was initially developed to treat B cell malignancies [4, 18]. Following initial success, Rituximab has also been approved for the management of rheumatoid arthritis and has undergone clinical trials demonstrating the successful use in a growing number of autoimmune diseases, including systemic lupus erythematosis, ITP, pemphigus, multiple sclerosis, and ANCA-associated vasculitis [19–23], in which dysregulated B cells are involved in the pathogenesis. A systematic review on the utilization of rituximab in ITP was published in 2007, included 313 patients from a total of 19 (eligible) studies and reported an overall response rate of 62.5% (as defined by a platelet increase to >50 × 109 cells/L) when given at a dose of 375 mg/m2 iv once weekly for 4 weeks . The median duration of the response was 10.5 months . More recent reports indicate that Rituximab may be more effective than various other immunosuppressants, particularly in the setting of refractory cases of ITP. Accordingly, Rituximab has been recognized as a useful drug in the second line of treatment for ITP either alone or in combination with corticosteroid, although it is not recommended as standard first-line therapy . However, whereas initial response rates are high, long-term follow up data on patients treated with Rituximab are sparse. It is still under investigation, how patients should be monitored and how frequently treatment should be repeated. In general, Rituximab is considered a relatively safe drug with only modest toxicity and infectious complications . Among side effects described are infusion-related reactions, serum sickness, and agranulocytosis [19, 26]. Due to the depletion of the B cell-mediated arm of the immune system, infections are a major concern. In different studies, the rate of serious infections has been between 2.8% to 45% (mean 12%) and the use of Rituximab as a direct cause of death has been estimated to 7% . These numbers may be difficult to interpret, since most of the patients were immunocompromised even prior to Rituximab treatment due to autoimmunity and the concomitant use of additional immunosuppressive agents. A recent multicenter retrospective study on the use of Rituximab in CVID-associated immune cytopenias reported a response rate of 85%, including 74% complete responses. However, after a mean follow-up time of 30 months after Rituximab, 10 out of the initial reponders relapsed and required re-treatment, and severe infections occurred in 24% .
Mechanism of action of Rituximab
Rituximab targets CD20 expressed by human B cells and a small subset of T cells [29, 30]. Within the B cell compartment, the large majority of B cells, with the exception of stem cells, pro-B cells, and plasma cells, express CD20 and therefore represent targets of Rituximab . Binding to CD20 by Rituximab induces B cell depletion by complement- and antibody-mediated cytotoxicity resulting in a 90% reduction in the number of CD20+ B cells in peripheral blood, lymphoid tissue, and bone marrow . The rationale behind using Rituximab in malignant and autoimmune diseases is therefore to eradicate malignant B cell clones or to prevent production of auto-antibodies by auto-reactive short-lived plasma cells derived from CD20 expressing B cells, respectively . Furthermore, several lines of evidence suggest that the T cell compartment may be modulated as well, including reduction of antigen-presenting cells to pathogenic auto-reactive T cells and modulation of the regulatory T cell compartment . Whereas transient depletion of peripheral B cells is common, Rituximab generally does not result in a decrease in immunoglobulin levels , a property that has been hypothesized to be attributable to the presence of long-lived plasma cells . However, low immunoglobulin levels can occur, and in rare cases Rituximab may result in low numbers of isotype-switched memory B cells . In a study involving Rituximab treatment of patients with rheumatoid arthritis, the incidence of decreased IgM was 40% whereas low IgG was seen in 3-6% of patients following the 5th cycle of Rituximab . In addition rare cases have been published describing profound hypogammaglobulinemia seven years after treatment for indolent lymphoma  as well as hypogammaglobulinemia with selective delayed recovery in memory B cells after adjuvant Rituximab treatment for NHL .
Previous cases of hypogammaglobulinemia associated with Rituximab treatment
Even if Rituximab only infrequently affects immunoglobulin levels, a central question is whether Rituximab may aggravate pre-existing CVID. Given that both CVID and Rituximab treatment result in loss of memory B cells and possibly affect the T cell population as well, it may be difficult to distinguish between the different origins of hypogammaglobulinemia. This issue was addressed by Diwakar et al., who described two patients presenting with aggravated immunodeficiency and bacterial infection requiring immunoglobulin infusions several months after Rituximab treatment for ITP , and therefore a very similar scenario to our case. This led the authors to suggest that Rituximab may have accelerated the presentation of immunodeficiency, since these patients had no notable infections prior to treatment but both experienced significant clinical deterioration, one of which was fatal, shortly after receiving Rituximab . In addition, one report describing persistent pan-hypogammaglobulinemia after Rituximab treatment for post-transplant EBV-associated AIHA may also represent Rituximab-induced aggravation of CVID .
So should Rituximab always be avoided in patients with ITP, in whom CVID has been diagnosed or is suspected? Several reports suggest that this may not be the case [39–42]. In the first case, a 34-year-old male with CVID complicated by inflammatory bowel disease, hepatic hyperplasia, splenomegaly, and portal hypertension, obtained a partial response for at least 11 months, and the authors were the first to suggest that treatment with Rituximab might be an option for patients with CVID and ITP refractory to other treatments (or for those in whom splenectomy is contraindicated) . Al-Ahmad et al. described the successful use of Rituximab in a patient with refractory ITP and CVID . This patient, a 36-year-old female with associated lymphadenopathy, EBV vireamia, and bronchiectatic changes received Rituximab for ITP and experienced a good response, even though she had a well-recognized CVID. In the next case concerning a 19-year-old woman with CVID complicated by granulomatous disease, neutropenia, and refractory ITP, a full response to Rituximab treatment was achieved with effect at 12 months in combination with low-dose steroid . Finally, El-Shanawany et al. described a case of a 65-year-old woman with CVID and severe recurrent thrombocytopenia despite treatment with high-dose IVIG and splenectomy, who had an excellent response to Rituximab therapy with disappearance of anti-platelet antibodies as reported 12 months after Rituximab . However, in none of these cases did hypogammaglobulinemia occur as a direct side effect to Rituximab, as these patients were all diagnosed with CVID prior to Rituximab and, notably, all of these patients received IVIG concomitantly with and after Rituximab treatment. In line with these case reports Gobert et al. described the results of a multicenter retrospective study concerning the use of Rituximab in 33 patients with CVID-associated immune cytopenias and found that Rituximab was associated with a subsequent decrease in residual IgG in individuals not receiving IgG replacement therapy . Furthermore, 24% of individuals developed severe infections after Rituximab treatment, and this was more frequently observed in those not receiving IgG replacement therapy. One splenectomized adult, who did not receive IgG replacement, died from S. pneumoniae pneumonia with sepsis 4 months after Rituximab treatment, underscoring the importance of addressing the potential development of severe hypogammaglobulinemia in patients with CVID treated with Rituximab.
Pathogenesis of the development of hypogammaglobulinemia in the presented patient
We initially considered whether this patient’s hypogammaglobulinemia might be a direct consequence of Rituximab treatment, which he had received about 21 months prior to his presentation with pneumococcal meningitis. However, based on his past medical history, which included treatment resistant ITP, splenomegaly, persistent lymphadenopathy, chronic gastrointestinal complaints, insufficient antibody responses to pneumococcal vaccination, and decreased IgA levels, we speculated that his ITP might rather be an autoimmune manifestation of an underlying CVID. This hypothesis was supported by the immunological characterization demonstrating a very low fraction of isotype-switched memory B cells (IgM- IgD- CD27+) in agreement with a diagnosis of CVID Freiburg class Ia. However, it should be noted that decreased numbers of isotype-switched memory B cells may rarely be secondary to Rituximab . Although we demonstrated neither TACI/ICOS mutations nor the absence of somatic hypermutation, CVID is a highly heterogeneous entity and these defects are only a few among several possible genetic abnormalities, which may be present in CVID. Based on the current knowledge on immunological effects of Rituximab, we do not believe that the concept of hypogammaglobulinemia arising as a direct effect of Rituximab sufficiently explains the case presented here. However, since this patient’s hypogammaglobulinemia became manifest and the frequency of infections increased following Rituximab infusions, it is possible that Rituximab was the factor that unmasked the full phenotype of an underlying CVID. In fact it was only after Rituximab treatment that he fulfilled the criteria defining CVID, in particular with respect to decreased IgG levels . Importantly, he did not have any condition that excludes the diagnosis of CVID. It seems that the immunodeficiency became more dominant than the autoreactivity (ITP), both present in severe cases of CVID, following Rituximab treatment.
Due to the raised fraction of CD3+ TCRαβ + CD4- CD8- double negative T cells in peripheral blood, the possibility of autoimmune lymphoproliferative syndrome (ALPS) was also considered. ALPS is characterized by benign lymphoproliferative disease and autoimmune cytopenia with a functional defect of T cells in FAS-induced apoptosis/CD95 signaling and therefore has considerable clinical and immunological overlap with CVID . However, in contrast to the case presented here, ALPS is generally characterized by hypergammaglobulinemia, although a minority of patients may present with hypogammaglobulinemia . ALPS patients also display a distinct pattern of biomarkers, including elevated soluble FasL and IL-10, which we did not find, and have a less pronounced decrease in isotype-switched memory B cells than CVID patients. Finally, we cannot exclude a possible minor effect of the patient’s splenectomy at age 11 on the distribution of lymphocyte subpopulations measured by flow cytometry on peripheral blood , although we do not find it likely to account for the significant abnormalities, including absence of isotype-switched memory B cells.