Marilia Cascalho, M.D., Ph.D.

Associate Professor of Surgery, Microbiology & Immunology
Accepting Students

Biography

Dr. Cascalho is a Physician -Scientist. She earned her MD in Lisbon, Portugal and her PhD at University of California in San Francisco. Dr. Cascalho’s interest on B cell biology was sparked by her early work on molecular mechanisms of immunoglobulin gene recombination and mutation (1-2). Her discovery that DNA mismatch repair drives B cell Ig gene hypermutation led her to investigate the genetic, molecular biological and cellular basis of immune fitness, viral evolution and oncogenesis. During the course of her work in somatic mutation and selection, Dr. Cascalho made an unexpected discovery, that B cell and immunoglobulin diversity promote T cell development and establishment of a diverse antigen receptor (TCR) repertoire. This observation led to current research directed at understanding how B cell and immunoglobulin diversity drive TCR diversification and the impact of such diversification on cellular immunity, in human health and disease and the relationship between T and B cell receptor diversity and immune fitness. The research also led to current inquiries into the genetic and molecular determinants of B cell responses to microorganisms and transplants. Dr. Cascalho also has a fundamental interest in the mechanisms that allow cells confronted by noxious extrinsic or intrinsic conditions (DNA double strand breaks and hypoxia in B cells and tumors, antibodies and complement in transplants) to survive.

(1) Cascalho M, Ma A, Lee S, Masat L, Wabl M. A quasi-monoclonal mouse. Science 272:1649-52, 1996.
(2) Cascalho M, Wong J, Steinberg C, Wabl M. Mismatch repair co-opted by hypermutation Science 279:1207-1210, 1998.

Research Interests

1. The control of immunity by the “transmembrane activator and CAML interactor” (TACI)

A. TACI variants in host defense In the development of immunity, what regulates genomic plasticity and selection on the one hand and phenotypic maturation on the other is not entirely known. However, these processes are clearly subject to rigorous control and when they fail, as in common variable immunodeficiency disease (CVID), severe clinical disease, including immunodeficiency, autoimmunity, lymphoproliferation and malignancy, ensues. Exploring the determinants of immune fitness and control Dr. Cascalho discovered that TACI, a receptor expressed by B cells and T cells, regulates expression of Blimp-1, the master transcription factor governing plasma cell differentiation (Mantchev et al., J Immunol.179:2282-2288, 2007; Tsuji et al., Blood, 118(22):5832-5839, 2011). Consistent with that function, mutants of TACI and related proteins are frequently associated with CVID (Barbosa et al., J Clin Immunol. 2014. 34:573-83). Most remarkable, however, Dr. Cascalho also discovered that while TACI-deficiency causes hypogammaglobulinemia, it does not prevent and indeed it promotes production in bursts of high affinity antibodies capable of protecting against enteric pathogens (Tsuji et al., J Clin Invest 2014. 124:4857-66). Thus, presence or absence of functional TACI does not determine immune competence but rather changes qualitatively how the immune system responds to environmental challenges. Because human variants of TACI that impair TACI function are frequent in healthy populations, this discovery raises the possibility that TACI variants might in fact be adaptations that confer protection against pathogens common in some populations.
Current research interrogates how certain TACI variants in a population contribute to the control of microbial spread. We hypothesize that TACI variants by changing the properties of innate and adaptive B cell responses alter microbial infectivity and dissemination through populations.

B. Genetic polymorphisms and outcome of alloimmunity in transplantation Our observations showing that TACI-deficiency (modeling the impact of dominant negative variants) changed rather than eliminated humoral immune responses, led us to ask if certain genetic TACI polymorphisms established the propensity to develop productive anti-donor antibody responses and antibody mediated rejection. We examined a cohort of kidney transplant recipients (198 alleles) who developed persistent anti-donor antibodies, or AMR detected in biopsies (with C4d deposition or with the morphologic properties pathognomonic of AMR) and a cohort of recipients who maintained healthy graft function for at least three years after transplantation (controls, 114 alleles). Our research shows that certain TACI polymorphisms were more frequent in recipients with DSA and/or rejection. For example, A173P, K188M, R189M, G190R, V220A, P251L and T234M were significantly more frequent in the rejecting population than in controls. Our results show that some alleles that are frequent in the general population (according to the ExAC database) almost exclusively partition in the rejecting group suggesting that common variants control immunity to transplantation. Current research addresses the question of how TACI variants increase the probability of antibody mediated rejection. In one possibility, TACI variants expressed in the heterozygotic state may alter the properties of anti-donor antibodies (DSA) produced in response to transplantation such as affinity/avidity and/ or isotype. These questions are addressed in murine models of transplantation and in recipients of living-donor kidney transplants.

C. Donor–specific B cell responses in transplantation. B cell responses to organ transplantation are now widely considered the most vexing biological hurdle to long-term success of organ transplants (Cascalho et al. Immunity 14:437-446, 2001; Cascalho et al. Journal of Immunology. 2013. 190 :875-9). How B cell responses eventuate in rejection in some recipients but not in others, and how B cell responses appear to protect some grafts and destroy others is not understood. Our research established that donor-specific B cell responses occur in most if not all transplant recipients, and that in most recipients these responses do not trigger acute rejection (Lynch et al. Am J Transplant. 13(7):1713-1723, 2013). Our findings showed that healthy graft outcomes (in non-sensitized recipients prior to transplantation) are not a consequence of absence of donor- specific responses, as it had been previously thought. Rather, donor–specific B cell responses may actually induce resistance of the graft to immune-injury, a condition known as accommodation, and perhaps tolerance (Dijke et al. Journal of Heart and Lung Transplantation. 2016. 35:704-10).
Our observations brought to the forefront the idea that some B cells comprising the donor-specific response may be beneficial towards the graft while others may be pathogenic and that the balance between pathogenic and protective responses may depend on TACI function. To distinguish between the two types of responses we undertake rigorous analysis of B cell clonal evolution, overtime, in recipients with TACI variants who developed rejection and in recipients expressing TACI-WT alleles that maintained healthy grafts up to one year after transplantation. Our preliminary findings indicate that donor specific B cell clonal evolution (analyzed by Ig H+L Sanger sequencing of single sorted B cells from the blood and by NGS of Ig H DNA obtained from core biopsies) distinguishes those with certain TACI alleles who undergo antibody-mediated rejection from those (with TACI-WT alleles) who maintain healthy graft function, long before donor-specific antibodies rise in the blood. Results show that antibody-mediated rejection is preceded (by several months) by an increase in the frequency of donor-specific IgG B cells and by extensive donor-specific IgG B cell intra-clonal diversification owing to somatic hypermutation and Ig affinity maturation. In contrast, in recipients that maintain healthy graft function, donor-specific IgG B cells do not increase in frequency with time after the peri-transplant period and clones do not seem to persist or mature. Instead the donor-specific B cell repertoire appears to be constantly changing suggesting deletion of donor-specific clones as they mature.

2. Ig diversification co-opted to enhance immunity to mutable microbes Dr. Cascalho’s interest in immunoglobulin gene diversification started during her graduate training in Dr. Wabl’s laboratory at UCSF. Using mice with targeted disruption of DNA mismatch repair enzymes Drs. Cascalho and Wabl discovered that DNA mismatch repair engenders plasticity, as mismatch repair enzymes are co-opted toward mutating the Ig genes (Cascalho et al. Science 272:1649-1652, 1996; Cascalho et al. Science 279:1207-1210, 1998). Later, as Assistant Professor of Immunology and Pediatrics at the Mayo Clinic, Dr. Cascalho and her team discovered that nuclear localization controls mismatch repair (Wu et al., Mol Cell Biol 23: 3320-3328, 2003) and that the function of activation-induced cytidine deaminase, the enzyme that initiates somatic hypermutation of Ig genes, is regulated by its association with the DNA protein kinase complex (Wu et al., J Immunol 174:934-941, 2005).
The concept that DNA mismatch repair functions as a mutator at the Ig loci (confirmed and extended by many others since our early report) established a principle that can be applied well beyond the functional responses of B cells to understand and potentially to control the evolution of viruses and tumors. For example, we found that somatic hypermutation diversifies microbial genes and generates variants of viral proteins identical to those found in patients with persistent virus (HIV) and that these variants might potentially evoke immunity (Balin and Cascalho, Nucleic Acids Research 38:1575-1582, 2010; Balin et al., Curr HIV Res 6:10-18, 2008). This finding originated an entirely novel concept of vaccine design, the “mutable vaccine” [US Patent No: US 7,776,321 B2]. The mutable vaccine departs from canonical approach to vaccine design, which stresses a priori identification of variants. The “mutable vaccine” is a DNA vaccine that encodes a microbial antigen that diversifies in B cells undergoing somatic hypermutation. The variants are secreted from plasma cells and in turn evoke variant-specific immunity in anticipation of variants generated naturally. Current research tests these concepts but already it has revealed that the vaccine can anticipate viral gene variants that would emerge in human populations (Cascalho et al. Immunotherapy. 2017). Current studies explore a novel double transgenic model the expresses and mutates the mutable vaccine conditionally to B cell activation and differentiation.

3. The control of immune checkpoint blockade by complement 3d (C3d) fragment in anti-tumor immunity
In the course of testing the mutable vaccine we made an unexpected observation. Because antigen with covalently attached C3d profoundly decreases the threshold for antibody responses, the mutable vaccine consists of DNA constructs that encode murine C3d attached to a viral antigen. While testing expression, diversification of the viral antigen and immunogenicity, the constructs were expressed in pre-B lymphoma cells introduced into isogeneic mice. Protective immunity was readily discerned as transfected lymphoma cells generated slowly growing tumors while mock transfected lymphoma cells generated rapidly growing tumors. What we did not anticipate, however, was that control lymphoma cells expressing only C3d as a free polypeptide and no viral antigen grew slower than mock-transfected lymphoma cells in isogeneic mice and 25% of the lymphomas spontaneously resolved. The impact of free C3d was not limited to lymphomas as expression of C3d in B16 melanoma cells also slowed tumor growth and prolonged survival of mice in which the cells were introduced. C3d, expressed as a free peptide, thus appeared to evoke resistance to tumors. Thus, free C3d, a fragment of the third component of complement, inside tumor cells or associated with irradiated tumor cells and unattached to antigen, recruits, accelerates and amplifies anti-tumor T-cell responses, allowing immunity to reverse or even to prevent tumor growth.
Despite expression of immunogenic polypeptides, tumors escape immune surveillance by engaging T cell checkpoint regulators and expanding regulatory T cells, among other mechanisms. What orchestrates these controls is unknown. Our findings revealed that C3d enhances anti-tumor immunity independently of B cells, NK cells or antibodies, but does so by increasing tumor infiltrating CD8-positive lymphocytes, by depleting regulatory T cells and by suppressing expression of programmed cell death protein 1 (PD-1) by T cells. These properties of C3d appear specific for the tumor, dependent on complement receptor 2 and incur no obvious systemic toxicity. The heretofore un-recognized properties of free C3d suggest that protein might determine the effectiveness of immune surveillance and that increasing availability of the protein might prove advantageous in the treatment or prevention of cancer and pre-malignant conditions. We are currently pursuing these avenues of inquiry. This research yielded a patent, recently converted [“C3d cellular and acellular vaccines for the prevention and treatment of cancer.” PCT NO:US-01/34733] and a manuscript. (Platt et al. J Clinical Investigation Insight. 2017)

Research Opportunities for Rotating Students

1. The control of immunity by the “transmembrane activator and CAML interactor” (TACI)

A. TACI variants in host defense
B. Genetic polymorphisms and outcome of alloimmunity in transplantation
C. Donor–specific B cell responses in transplantation.

2. Ig diversification co-opted to enhance immunity to mutable microbes

3. The control of immune checkpoint blockade by complement 3d (C3d) fragment in anti-tumor immunity

Publications