IgH sequences in common variable immune deficiency reveal altered B cell development and selection

Abstract

Common variable immune deficiency (CVID) is the most common symptomatic primary immune deficiency, affecting ~1 in 25,000 persons. These patients suffer from impaired antibody responses, autoimmunity, and susceptibility to lymphoid cancers. To explore the cellular basis for these clinical phenotypes, we conducted high-throughput DNA sequencing of immunoglobulin heavy chain gene rearrangements from 93 CVID patients and 105 control subjects and sorted naïve and memory B cells from 13 of the CVID patients and 10 of the control subjects. The CVID patients showed abnormal VDJ rearrangement and abnormal formation of complementarity-determining region 3 (CDR3). We observed a decreased selection against antibodies with long CDR3s in memory repertoires and decreased variable gene replacement, offering possible mechanisms for increased patient autoreactivity. Our data indicate that patient immunodeficiency might derive from both decreased diversity of the naïve B cell pool and decreased somatic hypermutation in memory repertoires. The CVID patients also exhibited an abnormal clonal expansion of unmutated B cells relative to the controls. Although impaired B cell germinal center activation is commonly viewed as causative in CVID, these data indicate that CVID B cells diverge from controls as early as the pro-B stage, cell and suggest possible explanations for the increased incidence of autoimmunity, immunodeficiency, and lymphoma CVID patients.

Fig. 1. CVID B cell repertoires show altered formation and selection of IgH

(A, left) Out-of-frame IgH sequences of CVID patients have shorter CDR3s than healthy controls (P=0.0128 for CVID Group I, P=0.00021 for CVID Group II). These sequences provide a measure of pre-selection VDJ gene rearrangement features. (A, middle) Productive but non-mutated IgH CDR3s are shorter than those of out-of-frame sequences. The difference between nonproductive and productive sequence CDR3 lengths in CVID patients is less than in controls, resulting in a productive unmutated repertoire with similar CDR3 lengths. (A, right) Productive IgH sequences that have undergone SHM have even shorter CDR3s on average but the selection for shorter sequences is significantly weaker in CVID Group I than in CVID Group II or healthy controls (P=0.0005). (B) Shorter CDR3s of out-of-frame sequences in CVID have fewer non-templated (N1 and N2) bases compared to healthy controls (P=0.0099 for CVID Group I, P<0.00001 for CVID Group II) and increased exonuclease digestion of segment ends compared to healthy controls (P=0.003 for CVID Group II). (C) Weakened selection for shorter CDR3s in CVID Group I than in healthy controls is also observed in naïve B cell IgM and IgD (P=0.0270 for IgM, P=0.0120 for IgD). (D) This weakened selection is also seen in mRNA sequences from sorted memory B cells in switched and unswitched isotypes (IgM, P=0.0020 for CVID Group I, P=0.0066 for CVID Group II; IgD, P=0.0020 for CVID Group I, P=0.0140 for CVID Group II; IgG1, P=0.0182 for CVID Group I; IgG2, P=0.0039 for CVID Group II; IgG3, P=0.0357 for CVID Group I, P=0.0406 for CVID Group II). All P-values are from the Wilcoxon-Mann-Whitney test.

Fig. 2. CVID subjects show decreased VH-replacement

(A) Bayesian modeling of the sequence characteristics present after VH-replacement shows low levels of VH-replacement in non-functional IgH sequences in both CVID patients and controls. (B) Naïve B cells show less evidence of VH-replacement in Group I CVID compared to healthy controls (IgM, P=0.0380; IgD, P=0.0120), while Group II CVID was comparable to healthy controls. (C) Mutated memory B cells show less VH-replacement in CVID than healthy controls for several isotypes and consistently decreased VH-replacement in CVID Group I over CVID Group II and healthy controls (IgM, P=0.0120 for CVID Group I, P=0.0326 for CVID Group II; IgD, P=0.0180 for CVID Group I, P=0.0127 for CVID Group II; IgG1, P=0.0364 for CVID Group I; IgG3, P=0.0213 for CVID Group II). All P-values are from the Wilcoxon-Mann-Whitney test.

Fig. 3. Naïve B cell repertoire richness is decreased in CVID

(A) The estimated lower bound of the unmutated IgH repertoire richness plotted against sequence read numbers per sample, both in log-scale. A linear regression model is shown for CVID Group I and II and healthy controls with the 95% confidence band. Both CVID groups show decreased richness compared to healthy controls. (B) Mutated IgH sequences show comparable richness in CVID and healthy controls. Repertoire richness estimates were calculated with the Chao2 estimator (see Methods).

Fig. 4. Somatic hypermutation levels are markedly decreased in CVID

(A) Productive IgH sequences from CVID subjects have fewer sequences with SHM compared to healthy controls (P<0.00001 for CVID Group I, P=0.00089 for CVID Group II). (B) The average percentage of mutated IGHV-gene nucleotides in B cells with productive IgH sequences showing evidence of mutation is significantly reduced in both CVID Group I and II compared to healthy controls (P<0.00001 for CVID Group I, P<0.00001 for CVID Group II). (C) A smaller fraction of CVID memory B cells show IgH SHM compared to healthy controls (IgM, P=0.0281 for CVID Group I, P=0.0471 for CVID Group II; IgG1, P=0.0226 for CVID Group I, P=0.0375 for CVID Group II; IgG2 P=0.0242 for CVID Group II; IgG3, P=0.0334 for CVID Group I). (D) Sorted memory B cells have consistently reduced levels of SHM in both groups of CVID compared to healthy controls across isotypes (IgM, P=0.0040 for CVID Group I, P=0.0021 for CVID Group II; IgD, P=0.0010 for CVID Group I, P=0.0028 for CVID Group II; IgA1 P=0.0076 for CVID Group II; IgA2 P=0.0209 for CVID Group II; IgG1, P=0.0182 for CVID Group I; IgG2 P=0.0296 for CVID Group II; IgG3, P=0.0357 for CVID Group I). All P-values are from the Wilcoxon-Mann-Whitney test.

Fig. 5. CVID subjects show altered B cell clonal expansions

(A) Contributions of clonally-expanded B cells to the repertoires of CVID and healthy controls are similar, as gauged by a ‘clonality score’ metric that is independent of sequencing depth. (B) CVID patients show a striking increase in the proportion of clonally-expanded B cells with germline IgH sequences, compared to healthy controls (P<0.00001 for CVID Group I, P<0.00001 for CVID Group II). P-values are from the Wilcoxon-Mann-Whitney test.

Fig. 6. Only a subset of CVID patients with lymphoma show elevated peripheral blood B cell clonality

(A) CVID patients without a history of lymphoma show similar clonality compared to healthy controls. (B) Some CVID Group I patients with a history of lymphoma show increased clonality of peripheral blood B cells.