Alex Gitlin, M.D., Ph.D. is currently an Instructor in the Department of Pathology at Stanford University School of Medicine. Prior to Stanford, Alex received his M.D. from Weill Cornell Medicine (2017) and his Ph.D. from Rockefeller University (2016) as part of the Medical Scientist Training Program (MSTP) at the Tri-Institutional M.D.-Ph.D. program. During his graduate training, Alex worked on the cellular and molecular mechanisms underlying germinal center reactions and the formation of long-lived humoral immunity. His work elucidated the mechanisms by which CD4+ T cells induce selective clonal expansion of germinal center B cells during the immune response. Currently, Alex's clinical and research interests lie in understanding the molecular mechanisms by which the innate immune system mounts inflammatory responses that are commensurate with the threat level posed to the host.
Honors & Awards
Harold M. Weintraub Graduate Student Award, Fred Hutchinson Cancer Research Center (2016)
Ruth L. Kirchstein National Research Service (F30), National Institutes of Allergy and Infectious Diseases, NIH (2014-2017)
Herman L. Jacobius Award for Excellence in Pathology, Weill Cornell Medicine (2017)
Medical Scientist Training Program, NIH (2009-2017)
Residency: Stanford University Department of Pathology (2020) CA
Medical Education: Weill Cornell Medical College (2017) NY
Ph.D., The Rockefeller University, Immunology (2016)
M.D., Weill Cornell Medicine, Medicine (2017)
A.B., Harvard College, Chemistry & Physics (2009)
Integration of innate immune signaling by caspase-8 cleavage of N4BP1.
Mutations in the death receptor Fas1,2 or its ligand FasL3 cause autoimmune lymphoproliferative syndrome (ALPS), whereas mutations in caspase-8 or its adaptor FADD - which mediate cell death downstream of Fas/FasL - cause severe immunodeficiency in addition to ALPS4-6. Mouse models have corroborated a role for FADD-caspase-8 in promoting inflammatory responses7-12, but the mechanisms underlying immunodeficiency remain undefined. Here, we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased production of select cytokines upon Toll-like receptor (TLR) 1/2, TLR7, or TLR9 stimulation, but not upon TLR3 or TLR4 engagement. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, impaired TLR3 and TLR4 cytokine responses of caspase-8-deficient macrophages13 were largely rescued by co-deletion of N4BP1. Thus, persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumor necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by FADD-caspase-8 mutations.
View details for DOI 10.1038/s41586-020-2796-5
View details for PubMedID 32971525
Independent Roles of Switching and Hypermutation in the Development and Persistence of B Lymphocyte Memory.
2016; 44 (4): 769–81
Somatic hypermutation (SHM) and class-switch recombination (CSR) increase the affinity and diversify the effector functions of antibodies during immune responses. Although SHM and CSR are fundamentally different, their independent roles in regulating B cell fate have been difficult to uncouple because a single enzyme, activation-induced cytidine deaminase (encoded by Aicda), initiates both reactions. Here, we used a combination of Aicda and antibody mutant alleles that separate the effects of CSR and SHM on polyclonal immune responses. We found that class-switching to IgG1 biased the fate choice made by B cells, favoring the plasma cell over memory cell fate without significantly affecting clonal expansion in the germinal center (GC). In contrast, SHM reduced the longevity of memory B cells by creating polyreactive specificities that were selected against over time. Our data define the independent contributions of SHM and CSR to the generation and persistence of memory in the antibody system.
View details for DOI 10.1016/j.immuni.2016.01.011
View details for PubMedID 26944202
View details for PubMedCentralID PMC4838502
HUMORAL IMMUNITY. T cell help controls the speed of the cell cycle in germinal center B cells.
Science (New York, N.Y.)
2015; 349 (6248): 643–46
The germinal center (GC) is a microanatomical compartment wherein high-affinity antibody-producing B cells are selectively expanded. B cells proliferate and mutate their antibody genes in the dark zone (DZ) of the GC and are then selected by T cells in the light zone (LZ) on the basis of affinity. Here, we show that T cell help regulates the speed of cell cycle phase transitions and DNA replication of GC B cells. Genome sequencing and single-molecule analyses revealed that T cell help shortens S phase by regulating replication fork progression, while preserving the relative order of replication origin activation. Thus, high-affinity GC B cells are selected by a mechanism that involves prolonged dwell time in the DZ where selected cells undergo accelerated cell cycles.
View details for DOI 10.1126/science.aac4919
View details for PubMedID 26184917
View details for PubMedCentralID PMC4809261
- Immunology: Fifty years of B lymphocytes. Nature 2015; 517 (7533): 139–41
Clonal selection in the germinal centre by regulated proliferation and hypermutation.
2014; 509 (7502): 637–40
During immune responses, B lymphocytes clonally expand and undergo secondary diversification of their immunoglobulin genes in germinal centres (GCs). High-affinity B cells are expanded through iterative interzonal cycles of division and hypermutation in the GC dark zone followed by migration to the GC light zone, where they are selected on the basis of affinity to return to the dark zone. Here we combine a transgenic strategy to measure cell division and a photoactivatable fluorescent reporter to examine whether the extent of clonal expansion and hypermutation are regulated during interzonal GC cycles. We find that both cell division and hypermutation are directly proportional to the amount of antigen captured and presented by GC B cells to follicular helper T cells in the light zone. Our data explain how GC B cells with the highest affinity for antigen are selectively expanded and diversified.
View details for DOI 10.1038/nature13300
View details for PubMedID 24805232
View details for PubMedCentralID PMC4271732
Squalene-based adjuvants stimulate CD8 T cell, but not antibody responses, through a RIPK3-dependent pathway.
The squalene-based oil-in-water emulsion (SE) vaccine adjuvant MF59 has been administered to more than 100 million people in more than 30 countries, in both seasonal and pandemic influenza vaccines. Despite its wide use and efficacy, its mechanisms of action remains unclear. In this study we demonstrate that immunization of mice with MF59 or its mimetic AddaVax (AV) plus soluble antigen results in robust antigen-specific antibody and CD8 T cell responses in lymph nodes and non-lymphoid tissues. Immunization triggered rapid RIPK3-kinase dependent necroptosis in the lymph node which peaked at 6 hours, followed by a sequential wave of apoptosis. Immunization with alum plus antigen did not induce RIPK3 kinase-dependent signaling. RIPK3-dependent signaling induced by MF59 or AV was essential for cross-presentation of antigen to CD8 T cells by Batf3-dependent CD8+ DCs. Consistent with this, RIPK3-kinase deficient or Batf3 deficient mice were impaired in their ability to mount adjuvant-enhanced CD8 T cell responses. However, CD8 T cell responses were unaffected in mice deficient in MLKL, a downstream mediator of necroptosis. Surprisingly, antibody responses were unaffected in RIPK3-kinase or Batf3 deficient mice. In contrast, antibody responses were impaired by in vivo administration of the pan-caspase inhibitor Z-VAD-FMK, but normal in caspase-1 deficient mice, suggesting a contribution from apoptotic caspases, in the induction of antibody responses. These results demonstrate that squalene-based vaccine adjuvants induce antigen-specific CD8 T cell and antibody responses, through RIPK3-dependent and-independent pathways, respectively.
View details for DOI 10.7554/eLife.52687
View details for PubMedID 32515732
ICAMs support B cell interactions with T follicular helper cells and promote clonal selection.
The Journal of experimental medicine
2017; 214 (11): 3435–48
The germinal center (GC) reaction begins with a diverse and expanded group of B cell clones bearing a wide range of antibody affinities. During GC colonization, B cells engage in long-lasting interactions with T follicular helper (Tfh) cells, a process that depends on antigen uptake and antigen presentation to the Tfh cells. How long-lasting T-B interactions and B cell clonal expansion are regulated by antigen presentation remains unclear. Here, we use in vivo B cell competition models and intravital imaging to examine the adhesive mechanisms governing B cell selection for GC colonization. We find that intercellular adhesion molecule 1 (ICAM-1) and ICAM-2 on B cells are essential for long-lasting cognate Tfh-B cell interactions and efficient selection of low-affinity B cell clones for proliferative clonal expansion. Thus, B cell ICAMs promote efficient antibody immune response by enhancement of T cell help to cognate B cells.
View details for DOI 10.1084/jem.20171129
View details for PubMedID 28939548
View details for PubMedCentralID PMC5679169
The microanatomic segregation of selection by apoptosis in the germinal center.
Science (New York, N.Y.)
2017; 358 (6360)
B cells undergo rapid cell division and affinity maturation in anatomically distinct sites in lymphoid organs called germinal centers (GCs). Homeostasis is maintained in part by B cell apoptosis. However, the precise contribution of apoptosis to GC biology and selection is not well defined. We developed apoptosis-indicator mice and used them to visualize, purify, and characterize dying GC B cells. Apoptosis is prevalent in the GC, with up to half of all GC B cells dying every 6 hours. Moreover, programmed cell death is differentially regulated in the light zone and the dark zone: Light-zone B cells die by default if they are not positively selected, whereas dark-zone cells die when their antigen receptors are damaged by activation-induced cytidine deaminase.
View details for DOI 10.1126/science.aao2602
View details for PubMedID 28935768
View details for PubMedCentralID PMC5957278
Design and crystal structure of a native-like HIV-1 envelope trimer that engages multiple broadly neutralizing antibody precursors in vivo.
The Journal of experimental medicine
2017; 214 (9): 2573–90
Induction of broadly neutralizing antibodies (bNAbs) by HIV-1 envelope glycoprotein immunogens would be a major advance toward an effective vaccine. A critical step in this process is the activation of naive B cells expressing germline (gl) antibody precursors that have the potential to evolve into bNAbs. Here, we reengineered the BG505 SOSIP.664 glycoprotein to engage gl precursors of bNAbs that target either the trimer apex or the CD4-binding site. The resulting BG505 SOSIP.v4.1-GT1 trimer binds multiple bNAb gl precursors in vitro. Immunization experiments in knock-in mice expressing gl-VRC01 or gl-PGT121 show that this trimer activates B cells in vivo, resulting in the secretion of specific antibodies into the sera. A crystal structure of the gl-targeting trimer at 3.2-Å resolution in complex with neutralizing antibodies 35O22 and 9H+109L reveals a native-like conformation and the successful incorporation of design features associated with binding of multiple gl-bNAb precursors.
View details for DOI 10.1084/jem.20161160
View details for PubMedID 28847869
View details for PubMedCentralID PMC5584115
Sequencing and cloning of antigen-specific antibodies from mouse memory B cells.
2016; 11 (10): 1908–23
Methods to identify genes encoding immunoglobulin heavy and light chains from single B lymphocytes vary in efficiency, error rate and practicability. Here we describe a protocol to sequence and clone the variable antibody region of single antigen-specific mouse memory B cells for antibody production. After purification, antigen-specific mouse memory B cells are first single-cell-sorted by fluorescence-activated cell sorting (FACS), and V(D)J transcripts are amplified by RT-PCR. Fragments are then combined with linearized expression vectors, assembled in vitro as part of a sequence- and ligation-independent cloning (SLIC) reaction and then transformed into Escherichia coli. Purified vectors can then be used to produce monoclonal antibodies in HEK293E suspension cells. This protocol improves the amplification efficiency of antibody variable genes and accelerates the cloning workflow. Antibody sequences will be available in 3-4 d, and microgram to milligram amounts of antibodies are produced within 14 d. The new protocol should be useful for addressing fundamental questions about antigen-specific memory B cell responses, as well as for characterizing antigen-specific antibodies.
View details for DOI 10.1038/nprot.2016.102
View details for PubMedID 27658009
HIV Vaccine Design to Target Germline Precursors of Glycan-Dependent Broadly Neutralizing Antibodies.
2016; 45 (3): 483–96
Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.
View details for DOI 10.1016/j.immuni.2016.08.016
View details for PubMedID 27617678
View details for PubMedCentralID PMC5040827
Sequential Immunization Elicits Broadly Neutralizing Anti-HIV-1 Antibodies in Ig Knockin Mice.
2016; 166 (6): 1445–58.e12
A vaccine that elicits broadly neutralizing antibodies (bNAbs) against HIV-1 is likely to be protective, but this has not been achieved. To explore immunization regimens that might elicit bNAbs, we produced and immunized mice expressing the predicted germline PGT121, a bNAb specific for the V3-loop and surrounding glycans on the HIV-1 spike. Priming with an epitope-modified immunogen designed to activate germline antibody-expressing B cells, followed by ELISA-guided boosting with a sequence of directional immunogens, native-like trimers with decreasing epitope modification, elicited heterologous tier-2-neutralizing responses. In contrast, repeated immunization with the priming immunogen did not. Antibody cloning confirmed elicitation of high levels of somatic mutation and tier-2-neutralizing antibodies resembling the authentic human bNAb. Our data establish that sequential immunization with specifically designed immunogens can induce high levels of somatic mutation and shepherd antibody maturation to produce bNAbs from their inferred germline precursors.
View details for DOI 10.1016/j.cell.2016.07.030
View details for PubMedID 27610569
View details for PubMedCentralID PMC5019122
Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice.
2016; 7: 10618
VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype.
View details for DOI 10.1038/ncomms10618
View details for PubMedID 26907590
View details for PubMedCentralID PMC4770077
Immunization for HIV-1 Broadly Neutralizing Antibodies in Human Ig Knockin Mice.
2015; 161 (7): 1505–15
A subset of individuals infected with HIV-1 develops broadly neutralizing antibodies (bNAbs) that can prevent infection, but it has not yet been possible to elicit these antibodies by immunization. To systematically explore how immunization might be tailored to produce them, we generated mice expressing the predicted germline or mature heavy chains of a potent bNAb to the CD4 binding site (CD4bs) on the HIV-1 envelope glycoprotein (Env). Immunogens specifically designed to activate B cells bearing germline antibodies are required to initiate immune responses, but they do not elicit bNAbs. In contrast, native-like Env trimers fail to activate B cells expressing germline antibodies but elicit bNAbs by selecting for a restricted group of light chains bearing specific somatic mutations that enhance neutralizing activity. The data suggest that vaccination to elicit anti-HIV-1 antibodies will require immunization with a succession of related immunogens.
View details for DOI 10.1016/j.cell.2015.06.003
View details for PubMedID 26091035
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Dynamic signaling by T follicular helper cells during germinal center B cell selection.
Science (New York, N.Y.)
2014; 345 (6200): 1058–62
T follicular helper (T(FH)) cells select high-affinity, antibody-producing B cells for clonal expansion in germinal centers (GCs), but the nature of their interaction is not well defined. Using intravital imaging, we found that selection is mediated by large but transient contacts between T(FH) and GC B cells presenting the highest levels of cognate peptide bound to major histocompatibility complex II. These interactions elicited transient and sustained increases in T(FH) intracellular free calcium (Ca(2+)) that were associated with T(FH) cell coexpression of the cytokines interleukin-4 and -21. However, increased intracellular Ca(2+) did not arrest TFH cell migration. Instead, T(FH) cells remained motile and continually scanned the surface of many GC B cells, forming short-lived contacts that induced selection through further repeated transient elevations in intracellular Ca(2+).
View details for DOI 10.1126/science.1257861
View details for PubMedID 25170154
View details for PubMedCentralID PMC4519234
HIV-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice.
Proceedings of the National Academy of Sciences of the United States of America
2013; 110 (41): 16538–43
Effective control of HIV-1 infection in humans is achieved using combinations of antiretroviral therapy (ART) drugs. In humanized mice (hu-mice), control of viremia can be achieved using either ART or by immunotherapy using combinations of broadly neutralizing antibodies (bNAbs). Here we show that treatment of HIV-1-infected hu-mice with a combination of three highly potent bNAbs not only resulted in complete viremic control but also led to a reduction in cell-associated HIV-1 DNA. Moreover, lowering the initial viral load by coadministration of ART and immunotherapy enabled prolonged viremic control by a single bNAb after ART was withdrawn. Similarly, a single injection of adeno-associated virus directing expression of one bNAb produced durable viremic control after ART was terminated. We conclude that immunotherapy reduces plasma viral load and cell-associated HIV-1 DNA and that decreasing the initial viral load enables single bNAbs to control viremia in hu-mice.
View details for DOI 10.1073/pnas.1315295110
View details for PubMedID 24043801
View details for PubMedCentralID PMC3799352
Fate mapping for activation-induced cytidine deaminase (AID) marks non-lymphoid cells during mouse development.
2013; 8 (7): e69208
The Aicda gene encodes Activation-Induced cytidine Deaminase (AID), an enzyme essential for remodeling antibody genes in mature B lymphocytes. AID is also responsible for DNA damage at oncogenes, leading to their mutation and cancer-associated chromosome translocation in lymphoma. We used fate mapping and AID(GFP) reporter mice to determine if AID expression in the mouse extends beyond lymphocytes. We discovered that AID(cre) tags a small fraction of non-lymphoid cells starting at 10.5 days post conception (dpc), and that AID(GFP+) cells are detectable at dpc 11.5 and 12.5. Embryonic cells are tagged by AID(cre) in the submandibular region, where conditional deletion of the tumor suppressor PTEN causes squamous papillomas. AID(cre) also tags non-lymphoid cells in the embryonic central nervous system. Finally, in the adult mouse brain, AID(cre) marks a small fraction of diverse neurons and distinct neuronal populations, including pyramidal cells in cortical layer IV.
View details for DOI 10.1371/journal.pone.0069208
View details for PubMedID 23861962
View details for PubMedCentralID PMC3704518
T follicular helper cell dynamics in germinal centers.
Science (New York, N.Y.)
2013; 341 (6146): 673–77
T follicular helper (T(FH)) cells are a specialized subset of effector T cells that provide help to and thereby select high-affinity B cells in germinal centers (GCs). To examine the dynamic behavior of T(FH) cells in GCs in mice, we used two-photon microscopy in combination with a photoactivatable fluorescent reporter. Unlike GC B cells, which are clonally restricted, T(FH) cells distributed among all GCs in lymph nodes and continually emigrated into the follicle and neighboring GCs. Moreover, newly activated T(FH) cells invaded preexisting GCs, where they contributed to B cell selection and plasmablast differentiation. Our data suggest that the dynamic exchange of T(FH) cells between GCs ensures maximal diversification of T cell help and that their ability to enter ongoing GCs accommodates antigenic variation during the immune response.
View details for DOI 10.1126/science.1241680
View details for PubMedID 23887872
View details for PubMedCentralID PMC3941467
Rif1 prevents resection of DNA breaks and promotes immunoglobulin class switching.
Science (New York, N.Y.)
2013; 339 (6120): 711–15
DNA double-strand breaks (DSBs) represent a threat to the genome because they can lead to the loss of genetic information and chromosome rearrangements. The DNA repair protein p53 binding protein 1 (53BP1) protects the genome by limiting nucleolytic processing of DSBs by a mechanism that requires its phosphorylation, but whether 53BP1 does so directly is not known. Here, we identify Rap1-interacting factor 1 (Rif1) as an ATM (ataxia-telangiectasia mutated) phosphorylation-dependent interactor of 53BP1 and show that absence of Rif1 results in 5'-3' DNA-end resection in mice. Consistent with enhanced DNA resection, Rif1 deficiency impairs DNA repair in the G(1) and S phases of the cell cycle, interferes with class switch recombination in B lymphocytes, and leads to accumulation of chromosome DSBs.
View details for DOI 10.1126/science.1230624
View details for PubMedID 23306439
View details for PubMedCentralID PMC3815530
Chromatin-targeting small molecules cause class-specific transcriptional changes in pancreatic endocrine cells.
Proceedings of the National Academy of Sciences of the United States of America
2012; 109 (14): 5364–69
Under the instruction of cell-fate-determining, DNA-binding transcription factors, chromatin-modifying enzymes mediate and maintain cell states throughout development in multicellular organisms. Currently, small molecules modulating the activity of several classes of chromatin-modifying enzymes are available, including clinically approved histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors. We describe the genome-wide expression changes induced by 29 compounds targeting HDACs, DNMTs, histone lysine methyltransferases (HKMTs), and protein arginine methyltransferases (PRMTs) in pancreatic α- and β-cell lines. HDAC inhibitors regulate several hundred transcripts irrespective of the cell type, with distinct clusters of dissimilar activity for hydroxamic acids and orthoamino anilides. In contrast, compounds targeting histone methyltransferases modulate the expression of restricted gene sets in distinct cell types. For example, we find that G9a/GLP methyltransferase inhibitors selectively up-regulate the cholesterol biosynthetic pathway in pancreatic but not liver cells. These data suggest that, despite their conservation across the entire genome and in different cell types, chromatin pathways can be targeted to modulate the expression of selected transcripts.
View details for DOI 10.1073/pnas.1201079109
View details for PubMedID 22434908
View details for PubMedCentralID PMC3325696
A dynamic T cell-limited checkpoint regulates affinity-dependent B cell entry into the germinal center.
The Journal of experimental medicine
2011; 208 (6): 1243–52
The germinal center (GC) reaction is essential for the generation of the somatically hypermutated, high-affinity antibodies that mediate adaptive immunity. Entry into the GC is limited to a small number of B cell clones; however, the process by which this limited number of clones is selected is unclear. In this study, we demonstrate that low-affinity B cells intrinsically capable of seeding a GC reaction fail to expand and become activated in the presence of higher-affinity B cells even before GC coalescence. Live multiphoton imaging shows that selection is based on the amount of peptide-major histocompatibility complex (pMHC) presented to cognate T cells within clusters of responding B and T cells at the T-B border. We propose a model in which T cell help is restricted to the B cells with the highest amounts of pMHC, thus allowing for a dynamic affinity threshold to be imposed on antigen-binding B cells.
View details for DOI 10.1084/jem.20102477
View details for PubMedID 21576382
View details for PubMedCentralID PMC3173244